Proteomic analysis of the response of murine bone marrow derived macrophages to IFN y stimulation and infection with staphylococcus aureus

Proteomic analysis of the response of murine bone marrow derived macrophages to IFN-γ stimulation and infection with Staphylococcus aureus Inauguraldissertation zur Erlangung des akademischen Grades Doktor rerum naturalium (Dr rer nat.) die Mathematisch-Naturwissenschaftliche Fakultät der Ernst-Moritz-Arndt-Universität Greifswald Vorgelegt von Dinh Hoang Dang Khoa geboren am 12.06.1981 in Binh Thuan - Vietnam Greifswald, July 2010 i Dekan: Gutachter 1: Gutachter 2: Tag der Promotion: ii Content Abbreviations i List of Figures and Tables iii Summary 1 Introduction 1.1 Macrophages 1.1.1 Macrophage origin and morphology 1.1.2 Immunological function of macrophages 1.1.2.1 Microbial pathogen phagocytosis 1.1.2.2 Antigen presentation 1.1.2.3 Immune modulation 1.1.3 Other functions of macrophages 1.2 IFN gamma activation of macrophages 1.2.1 IFN gamma 1.2.2 Effects of IFN-γ on macrophages 1.3 Proteomics studies of macrophages 1.3.1 Strategies of proteomics analysis 1.3.2 Macrophage proteomics 11 1.4 Interaction of Staphylococcus aureus and macrophages 12 1.5 A reproducible experimental system - Bone marrow derived macrophages in serum-free culture 14 Materials and Methods 15 2.1 Materials 15 2.1.1 Chemicals 15 2.1.2 Instruments 16 2.1.3 Software 17 2.2 Methods 17 2.2.1 Sample preparation 17 2.2.1.1 Stem cell preparation, cultivation, and differentiation to macrophages 17 2.2.1.2 Interferon-γ activation of bone marrow derived macrophages 18 iii 2.2.1.3 S aureus infection 18 2.2.1.4 BMM protein extraction for proteome analysis 19 2.2.1.5 Determination of protein concentration 19 2.2.2 2D-DIGE approach 19 2.2.2.1 CyDye labeling reaction for DIGE experiment 19 2.2.2.2 Rehydration 20 2.2.2.3 IEF separation 20 2.2.2.4 Equilibration 21 2.2.2.5 Second dimension separation 21 2.2.3 Protein spot visualization 21 2.2.3.1 CyDye DIGE scanning 21 2.2.3.2 Colloidal coomassie staining 22 2.2.4 Spot detection and quantification 22 2.2.5 Mass spectrometry analysis 23 2.2.5.1 MALDI-TOF-MS/MS 23 2.2.5.1.1 Preparative gels 23 2.2.5.1.2 Protein identification by MALDI-TOF/TOF MS 23 2.2.5.2 Quantitative LC-MS/MS analysis 24 2.2.6 Functional classification of proteins 26 2.2.7 Transcriptomic analysis 26 Results 28 3.1 2-DE protein reference map of BMMs 30 3.2 IFN-γ effect on BALB/c and C57BL/6 macrophages 36 3.2.1 IFN-γ regulated proteins identified by 2D-DIGE technique 37 3.2.2 IFN-γ regulated proteins identified by LC-MS/MS and comparison with transcriptomic results 45 3.3 Comparative proteome analysis of BALB/c and C57BL/6 macrophages 59 3.3.1 Differences in proteomic profiles of BMM-BALB/c and BMM-C57BL/6 identified with the 2D – DIGE technique 60 3.3.2 Comparison of LC-MS/MS and transcriptomic data 64 iv 3.4 Effects of S aureus infection on the proteome pattern of IFN-γ stimulated BMMC57BL/6 70 Discussion 79 4.1 2-DE proteome reference map of bone marrow derived macrophages 79 4.2 IFN-γ effects on BMM-BALB/c and BMM-C57BL/6 identified by proteomic 2D-DIGE and LC-MS/MS approaches 80 4.2.1 Transcription regulation 81 4.2.2 p47 and p65 GTPases 82 4.2.3 Antigen presentation 83 4.2.4 Metabolism 85 4.2.5 Cell survival 86 4.2.6 Secretion of cathepsin L and metalloelastase 87 4.2.7 Well known, immunologically important proteins not influenced by IFN-γ treatment 88 4.3 Changes in the proteome of IFN-γ stimulated BMM-C57BL/6 due to S aureus infection 88 4.3.1 Anti-microbial proteins 88 4.3.2 Inflammatory regulation proteins 89 4.3.3 Cell-cell interaction 91 4.3.4 Metabolism 91 4.3.4.1 Protein and glucose uptake 91 4.3.4.2 Lipid metabolism 92 4.3.4.3 Cellular iron homeostasis 92 4.3.5 Immune-responsive gene protein 94 4.4 Differences in proteome of BMMs derived from strain BALB/c and C57BL/6 94 Conclusion 96 References 97 Affidavit Curriculum Vitae Acknowledgments Supplements v Abbreviations 2-DE : Two dimensional gel electrophoresis 2D-DIGE : Two-dimensional difference gel electrophoresis ACN : Acetonitrile APS : Ammonium persulphate BMM : Bone marrow derived macrophages CD : Cluster of differentiation CyDye : CyDye DIGE fluorescent dyes Da : Dalton DCs : Dendritic cells DIGE : Fluorescence difference gel electrophoresis DNA : Deoxyribonucleic acid DTT : Dithiothreitol ER : Endoplasmic reticulum FBS : Fetal bovine serum FCS : Fetal calf serum Fig : Figure HPLC : High performance liquid chromatography IAA : Iodoacetamide ID(s) : Identifier(s) IEF : Isoelectric focusing IFNGR : Interferon gamma receptor IFN-γ : Interferon gamma IL Interleukin iNOS : Inducible nitric oxide synthase IPG : Immobilized pH gradient IPI : International Protein Index kDa : Kilodalton i LC : Liquid chromatography LC-MS/MS : Liquid Chromatography-Tandem Mass Spectrometry LPS : Lipopolysaccharide MALDI : Matrix-assisted laser desorption/ionization MHC : Major histocompatibility complex : Minute Mr : Molecular mass mRNA : Messenger ribonucleic acid MS : Mass spectrometry MS/MS : Tandem mass spectrometry NADPH : Nicotinamide adenine dinucleotide phosphate NCBI : National Center for Biotechnology Information NK : Natural killer cell NO : Nitric oxide NOS : Nitric oxide synthase PANTHER : Protein Analysis Through Evolutionary Relationships PBS : Phosphate buffered saline PCA : Principal components analysis pI : Isoelectric point PTM : Post-translational modification RNA : Ribonucleic acid RNI : Reactive nitrogen intermediate ROI : Reactive oxygen intermediate ROS : Reactive oxygen species S aureus : Staphylococcus aureus SDS : Sodium dodecyl sulphate SDS-PAGE : Sodium dodecyl sulfate polyacrylamide gel electrophoresis STAT : Signal transducer and activator of transcription Suppl : Supplements TBS : TRIS-buffered saline ii TEMED : N,N,N',N'-tetramethylethylenediamine TGF-β : Transforming growth factor beta TH : T helper cell TLR : Toll-like receptor TNF : Tumor necrosis factor Tris : Tris(hydroxymethyl) aminomethane vs : versus List of Figures and Tables Figures Figure 1: Typical appearance of macrophage Figure 2: Overiew of BMMs proteomics and transcriptomics analyses 29 Figure 3: Molecular mass – isoelectric point plot 32 Figure 4: 2-DE proteome reference map of BMMs 33 Figure 5: Functional classification of identified proteins on 2-DE proteomic reference map 34 Figure 6: 2D-DIGE experiment scheme 38 Figure 7: Representative gel image of the IFN-γ effects on proteome of BMM-BALB/c 40 Figure 8: Representative gel image of the IFN-γ effects on proteome of BMM-C57BL/6 41 Figure 9: Induction of cathepsin B and cathepsin S protein isoforms due to IFN-γ stimulation 44 Figure 10: Principal component analysis of proteomic LC-MS/MS and transcriptomic data 47 Figure 11: Ratio plot of identified IFN-γ regulated genes and proteins 50 Figure 12: Functional classification of IFN-γ regulated proteins and genes identified by proteomic LCMS/MS and transcriptomic technique 52 Figure 13: Overlay between identified genes and proteins 53 Figure 14: mRNA and protein level of thirdteen immune related genes 55 Figure 15: Representative gels of differences in 2-DE protein expression profiles of BMM-BALB/c and BMM-C57BL/6 61 Figure 16: Different distribution of protein isoforms of BGLR and ERP29 in BMM-BALB/c and BMMC57BL/6 63 Figure 17: Ratio plot of mRNAs and proteins being present at different levels in a strain-dependent manner 66 Figure 18: Functional classification and cellular localization of the 343 proteins identified as different levels in BMM-BALB/c and BMM-C57BL/6 69 Figure 19: Experimental setting for identifying IFN-γ effects and S aureus effects in BMM-C57BL/6 71 iii Figure 20: Mapping of IFN-γ regulated genes/proteins and S aureus regulated proteins in BMM-C57BL/6 73 Figure 21: Functional classification of S aureus regulated proteins 76 Figure 22: Time-resolved analysis of the intensity changes of some selected proteins influenced by infection with S aureus 78 Tables Table 1: BMM batches used in the study 18 Table 2: The serial dilution of BSA-standard solution 19 Table 3: IEF program for Immobiline DryStrip pH 4-7, 24 cm 21 Table 4: Protein distribution on 2D proteomic reference map 31 Table 5: IFN-γ modulated protein spots in BMM-BALB/c and BMM-C57BL/6 identified by the 2D-DIGE technique 39 Table 6: Proteins identified in IFN-γ modulated protein spots 43 Table 7: Summary of genes and proteins identified by transcriptomic and proteomic LC-MS/MS technique as IFN-γ regulated 48 Table 8: Overlay of genes and proteins influenced by IFN-γ treatment 54 Table 9: Functions of immune related genes for which total mRNA and protein amount were not influenced by IFN-γ stimulation 55 Table 10: List of a total of 69 IFN-γ regulated proteins in BMM-BALB/c and/or BMM-C57BL/6 identified by LC-MS/MS technique 56 Table 11: Twenty seven genes for which changes by IFN-γ stimulation were observed at both transcriptional and translational level 58 Table 12: Immune related proteins which were not changed in total amount due to IFN-γ stimulation 58 Table 13: Protein spots identified by 2D-DIGE technique and displaying strain-specific differences in intensity 60 Table 14: Summary of genes and proteins displaying strain specific expression levels identified by transcriptomics and LC-MS/MS techniques 65 Table 15: Overlay of genes and proteins showing different expression or levels 67 Table 16: Proteins regulated by infection with S aureus in IFN-γ stimulated BMM-C57BL/6 72 Table 17: Proteins influenced in abundance by infection with S aureus at h and/or 24 h post infection 74 Table 18: Proteins influenced in abundance by infection with S aureus and IFN-γ stimulation 75 iv Summary Dissertation Summary Macrophages which are distributed throughout the normal body provide the first line of defence against microbial pathogen infections With vigorous phagocytosis ability, macrophages can eliminate a wide variety of invading microorganisms including viruses, bacteria, fungi and protozoa Macrophages also function as professional antigen presenting cells which connect innate and adaptive arms of the immune system Moreover, many secreted cytokines from macrophages are involved in modulation of the immune response IFN-γ is well known as a main macrophage stimulator IFN-γ stimulated macrophages possess higher bactericidal capacity than in normal state Many physiological and functional changes in IFN-γ stimulated macrophages were reported such as inducing in production of reactive oxygen species (ROI), nitric oxide (NO), and secretion of pro-inflammatory cytokines However, information about the changes in the proteome of macrophages upon activation by IFN-γ is still limited Murine bone marrow derived macrophages (BMMs) are a good model for investigating macrophage biology In this study, murine BMMs were generated from a well defined standardized serum-free culture system which ensures in comparison to established serumcultivation improved reproducibility and accuracy of the results Effects of stimulation with IFNγ on the proteome of BMMs from an infection-susceptible mouse strain BALB/c and a resistance mouse strain C57BL/6 were studied by complementary 2D-DIGE (gel-based) and LC-MS/MS (gel-free) approaches A 2-DE proteome reference map of BMMs was created from protein pools of BMMBALB/c and BMM-C57BL/6 proteins via 2-DE electrophoresis and MALDI-TOF/TOF-MS This reference map covers 252 identified protein spots of 145 unique proteins Functional analysis showed that “protein metabolism and modification”, “immunity and defense”, “cell structure and motility” were the most abundant biological functional groups among the identified proteins Applying the 2D-DIGE technique, we identified 18 and 19 proteins spots, respectively, for which spot intensities were significantly changed in BMM-BALB/c and BMM-C57BL/6 due to IFN-γ stimulation While LC-MS/MS analysis revealed 45 and 53 IFN-γ affected proteins, respectively, in BMM-BALB/c and BMM-C57BL/6 Interestingly, results of the two proteomics analyses showed that BMMs derived from susceptible strain BALB/c and resistance strain C57BL/6 responded to IFN-γ stimulation with a consistent pattern The functions of the identified IFN-γ regulated proteins could be assigned to transcription regulation (STAT1), microbicidal activity (members of p47 and p65 GTPases), antigen presentation (components of MHC class I and class II molecule, TAP2, lysosomal cathepsins), cell survival (PRDX4, NAMPT, AIF-1), and metabolism (hexokinases, ACSL1) 992* 711*' 988* 852* 864* 781* 761* 461*' 820*' 974' 938*' 785*' 983 959*' 911' 982* 973*' 962* 927*' 1003* 931* 502' 846' 830*' 204 1004 975*' 1009* 1052* 768* 556*' 71 94 104' 772*' 751*' 801*' 786* 668 89 106 512' 491 1023* 73 74* 101 1* 695* 777*' 14* 1018* 901*' 808*' 752*' 703* 648*' 541 1042 494* 346* 258*' 1043 1024* 895*' 685 603' 5* 1065 653* 1025* 1047*' 1045' 1046 1029*' 581* 1050*' 511' 385* 319* 1030*' 948' 713 738*' 1031 1049' 473' 26 121* 1060* 125* 96 90 192 265 257*' 263*' 264*' 270 164 256*' 384*' 444* 564*' 524*' 600* 472* 449*' 317*' 217 213 344 198 44 184* 175* 177*' 85' 747*' 538* 536*' 514*' 499*' 554* 779*' 577' 553 505*' 481 205* 182* 46* 216*197* 32* 103' 157 69 443* 134 62 894*' 783*' 579 664' 66* 311* 407*' 509* 776*' 469*' 244' 241*' 886*' 799*' 732*' 774 917 996* 960' 945*' 671* 566*' 174 65* 99* 250*' 288* 92 417* 83* 400* 119 377* 181' 169*' 381' 811*' 717' 656*' 601 432*' 715 765*' 748 670* 574*' 1002 754' 652*' 139* 172*' 168 454*' 166*' 151 708*' 578*' 453' 466' 807* 828*' 679* BALB/c + IFN-γ C57BL/6 + IFN-γ Dissertation A11 Figure A.3: Representative gel image BMM-BALB/c versus BMM-C57BL/6 in IFN-γ treatment condition Representative overlay image of 2D protein expression patterns of the IFN-γ stimulated BMM-BALB/c (green colour) and IFN-γ stimulated BMM-C57BL/6 (red colour) 204 protein spots identified at different levels in IFN-γ stimulated BMM-BALB/c and IFN-γ stimulated BMM-C57BL/6 are labeled with spot number 91 protein spots were successfully identified with MALDI-MS/MS Spot colour indicates the different in protein abundance between to mouse strains: red = higher in IFN-γ stimulated BMM-C57BL/6; green = lower in IFN-γ stimulated BMMC57BL/6; yellow = equal between IFN-γ stimulated BMM-BALB/c and IFN-γ stimulated BMM-C57BL/6 Higher- and lower-existing protein spots in IFN-γ stimulated BMM-C57BL/6 can be also distinguished by white and green letters, respectively Symbol * indicate protein spots which identified at different levels in BMM-BALB/c and BMM-C57BL/6 in both control and IFN-γ treatment condition Symbol ’ indicates successfully identified proteins spots with MALDI-MS/MS Supplements Supplements Accession Number CLH_MOUSE PKHO2_MOUSE AL3B1_MOUSE METK2_MOUSE ACTG_MOUSE CATD_MOUSE CSN4_MOUSE PON3_MOUSE ALDH2_MOUSE SFPQ_MOUSE DHB8_MOUSE EXOS6_MOUSE CATD_MOUSE OSTF1_MOUSE VATA_MOUSE MA2B1_MOUSE PRDX6_MOUSE ALDH2_MOUSE NDUV2_MOUSE 1433Z_MOUSE SNX5_MOUSE ATPA_MOUSE THIO_MOUSE ITAM_MOUSE MYOF_MOUSE COR1A_MOUSE IIGP1_MOUSE Gene entrez ID 67300 102595 67689 232087 11465 13033 26891 269823 11669 71514 14979 72544 13033 20409 11964 17159 11758 11669 72900 22631 69178 11946 22166 16409 226101 12721 60440 Protein Name Clathrin heavy chain Pleckstrin homology domain-containing family O member Aldehyde dehydrogenase family member B1 S-adenosylmethionine synthetase isoform type-2 Actin, cytoplasmic Cathepsin D COP9 signalosome complex subunit Serum paraoxonase/lactonase Aldehyde dehydrogenase, mitochondrial Splicing factor, proline- and glutamine-rich Estradiol 17-beta-dehydrogenase Exosome complex exonuclease MTR3 Cathepsin D Osteoclast-stimulating factor Vacuolar ATP synthase catalytic subunit A Lysosomal alpha-mannosidase Peroxiredoxin-6 Aldehyde dehydrogenase, mitochondrial NADH dehydrogenase [ubiquinone] flavoprotein 2, mitochondrial 14-3-3 protein zeta/delta Sorting nexin-5 ATP synthase subunit alpha, mitochondrial Thioredoxin Integrin alpha-M Myoferlin Coronin-1A Interferon-inducible GTPase Fold change (‘C57BL/6' /'BALB/c') -1.92 -1.92 2.51 2.51 1.56 1.56 -1.62 -1.62 1.82 1.82 1.58 1.58 1.58 4.48 4.48 -3.10 -3.10 -1.83 -1.83 -1.56 -1.56 -1.53 -1.53 Fold change (‘C57BL/6+IFN' /'BALB/c+IFN') -1.91 -1.91 -1.59 -1.59 2.42 2.42 1.63 1.63 -1.83 -1.83 3.13 3.13 8.71 8.71 -2.73 -2.73 -1.62 -1.62 -1.61 -1.61 - Dissertation 191.4 53.8 52.3 43.7 41.8 44.9 46.3 39.3 56.5 75.4 26.6 28.4 44.9 23.8 68.3 114.5 24.9 56.5 27.3 27.8 46.8 59.7 11.7 127.4 233.2 51.0 47.5 MW (kDa) 5.48 5.4 7.5 6.02 5.31 6.71 5.57 5.44 7.53 9.45 6.1 5.87 6.71 5.46 5.42 8.3 5.71 7.53 4.73 6.19 9.22 4.8 6.87 5.83 6.05 Protein pI Table A.2: Thirteen protein spots identified as different level in BMM-BALB/c and BMM-C57BL/6 which contained more than protein/spot Spot 250* 250* 512 512 536* 536* 556* 556* 648* 648* 738* 738* 753 753 753 808* 808* 830* 830* 894* 894* 974 974 1021 1021 1034 1034 The 13 protein spots contained total 25 proteins These proteins were identified as different levels between two mouse strains and contained two proteins/spot Symbol * indicate protein spots which identified at different levels in BMM-BALB/c and BMM-C57BL/6 in both control and IFN-γ treatment condition Positive fold change value means higher in BMM-C57BL/6, negative value means lower in BMM-C57BL/6 A12 Dissertation ACTB_MOUSE ACTB_MOUSE ACTG_MOUSE ACTG_MOUSE ACTG_MOUSE ACTG_MOUSE ACTG_MOUSE ACTG_MOUSE BGLR_MOUSE BGLR_MOUSE BGLR_MOUSE BGLR_MOUSE BGLR_MOUSE BGLR_MOUSE ERP29_MOUSE ERP29_MOUSE VIME_MOUSE VIME_MOUSE VIME_MOUSE VIME_MOUSE VIME_MOUSE VIME_MOUSE VIME_MOUSE VIME_MOUSE VIME_MOUSE VIME_MOUSE Accession Number 11461 11461 11465 11465 11465 11465 11465 11465 110006 110006 110006 110006 110006 110006 67397 67397 22352 22352 22352 22352 22352 22352 22352 22352 22352 22352 Gene entrez ID Actin, cytoplasmic Actin, cytoplasmic Actin, cytoplasmic Actin, cytoplasmic Actin, cytoplasmic Actin, cytoplasmic Actin, cytoplasmic Actin, cytoplasmic Beta-glucuronidase Beta-glucuronidase Beta-glucuronidase Beta-glucuronidase Beta-glucuronidase Beta-glucuronidase Endoplasmic reticulum protein ERp29 Endoplasmic reticulum protein ERp29 Vimentin Vimentin Vimentin Vimentin Vimentin Vimentin Vimentin Vimentin Vimentin Vimentin Protein Name Fold change (‘C57BL/6' /'BALB/c') -1.76 -2.09 -1.61 -1.50 -1.53 -2.08 -5.06 3.21 -2.33 1.86 1.72 4.90 -11.40 -1.82 -3.07 -2.95 2.21 2.42 1.92 2.51 1.68 Fold change (‘C57BL/6+IFN' /'BALB/c+IFN') -2.06 -2.90 -1.84 -1.65 -1.72 -1.82 -2.10 -1.80 -4.91 3.48 -2.27 1.94 2.12 5.57 -9.06 -2.42 -2.04 -2.06 -3.31 -3.81 1.58 1.89 2.03 41.7 41.7 41.8 41.8 41.8 41.8 41.8 41.8 74.2 74.2 74.2 74.2 74.2 74.2 28.8 28.8 53.7 53.7 53.7 53.7 53.7 53.7 53.7 53.7 53.7 53.7 MW (kDa) 5.29 5.29 5.31 5.31 5.31 5.31 5.31 5.31 6.16 6.16 6.16 6.16 6.16 6.16 5.9 5.9 5.06 5.06 5.06 5.06 5.06 5.06 5.06 5.06 5.06 5.06 Protein pI A13 The 25 protein spots which were identified as different levels in BMM-BALB/c and BMM-C57BL/6 contained proteins Each of proteins had some isoforms existed in higher levels in BMMs of one strain, however some other of it isoform existed in higher levels in the other strain Symbol * indicate protein spots which identified at different levels in BMM-BALB/c and BMM-C57BL/6 in both control and IFN-γ treatment condition Positive fold change value means higher in BMM-C57BL/6, negative value means lower in BMM-C57BL/6 886* 945* 566* 571 577 717 776* 846 256* 257* 258* 263* 264* 317* 751* 752* 381 432* 453 454* 461* 574* 578* 589 828* 829 Spot Table A.3: Five proteins of which total amount were equal, however their protein isoforms were differently distributed in two mouse strains Supplements Supplements Accession Number 1433F_MOUSE 1433Z_MOUSE ARP3_MOUSE ALDH2_MOUSE ENOA_MOUSE ANXA1_MOUSE ANXA5_MOUSE CATB_MOUSE CATD_MOUSE CATD_MOUSE CATD_MOUSE CATS_MOUSE CATS_MOUSE CATS_MOUSE CATZ_MOUSE COR1A_MOUSE CNDP2_MOUSE ERAP1_MOUSE HA12_MOUSE HA12_MOUSE HSP7C_MOUSE HSP7C_MOUSE HS90B_MOUSE ITAM_MOUSE PPGB_MOUSE PRDX1_MOUSE PRDX6_MOUSE PDIA6_MOUSE PDIA6_MOUSE TPM3_MOUSE Gene entrez ID 22629 22631 74117 11669 13806 16952 11747 13030 13033 13033 13033 13040 13040 13040 64138 12721 66054 80898 14964 14964 15481 15481 15516 16409 19025 18477 11758 71853 71853 59069 Protein Name 14-3-3 protein eta 14-3-3 protein zeta/delta Actin-related protein Aldehyde dehydrogenase, mitochondrial Alpha-enolase Annexin A1 Annexin A5 Cathepsin B Cathepsin D Cathepsin D Cathepsin D Cathepsin S Cathepsin S Cathepsin S Cathepsin Z Coronin-1A Cytosolic non-specific dipeptidase Endoplasmic reticulum aminopeptidase H-2 class I histocompatibility antigen, D-D alpha chain H-2 class I histocompatibility antigen, D-D alpha chain Heat shock cognate 71 kDa protein Heat shock cognate 71 kDa protein Heat shock protein HSP 90-beta Integrin alpha-M Lysosomal protective protein Peroxiredoxin-1 Peroxiredoxin-6 Protein disulfide-isomerase A6 Protein disulfide-isomerase A6 Tropomyosin alpha-3 chain Fold change (‘C57BL/6' /'BALB/c') -1.61 -1.66 -1.63 -1.73 -1.72 2.46 2.34 1.73 1.70 2.02 2.15 1.63 -1.77 -1.62 -143.06 -5.56 -1.53 -1.89 -1.79 -2.09 1.54 -2.15 -10.19 -1.50 Table A.4: Twenty three proteins of which total amount might be significant different in two strains Spot 765* 754 505* 901* 1049 603 656* 741 747* 777* 975* 772* 783* 799* 666 384* 407* 103 499* 514* 241* 244 169* 1051 895* 859 801* 652* 1045 708* Fold change (‘C57BL/6+IFN' /'BALB/c+IFN') -1.66 -1.53 -1.86 -2.51 -1.54 -1.71 -1.74 2.49 2.37 1.72 1.57 1.84 2.27 -2.14 -1.55 1.65 -100.70 -3.64 -1.62 -1.63 -2.11 -1.76 -2.26 -7.48 -1.57 -1.87 MW (kDa) 4.81 4.73 5.61 7.53 6.37 6.97 4.83 5.57 6.71 6.71 6.71 6.51 6.51 6.51 6.13 6.05 5.43 5.77 6.22 6.22 5.37 5.37 4.97 6.87 5.56 8.26 5.71 5 4.68 Protein pI Dissertation 28.2 27.8 47.3 56.5 47.1 38.7 35.7 37.3 44.9 44.9 44.9 38.4 38.4 38.4 34.0 51.0 52.7 106.5 41.1 41.1 70.8 70.8 83.3 127.4 53.8 22.2 24.9 48.1 48.1 32.8 Each of 23 proteins had some but not all of identified isoforms existed in higher levels in BMMs of one mouse strain Symbol * indicate protein spots which identified at different levels in BMM-BALB/c and BMM-C57BL/6 in both control and IFN-γ treatment condition Positive fold change value means higher in BMM-C57BL/6, negative value means lower in BMM-C57BL/6 A14 OST48_MOUSE EF1B_MOUSE IF5A1_MOUSE LEG1_MOUSE SAP3_MOUSE GNPI1_MOUSE HS90A_MOUSE HCLS1_MOUSE HCLS1_MOUSE ROA3_MOUSE HNRPF_MOUSE IVD_MOUSE LONM_MOUSE LONM_MOUSE NDUS3_MOUSE CNPY2_MOUSE PDPR_MOUSE GDIB_MOUSE GDIR2_MOUSE RHG25_MOUSE RINI_MOUSE SPA3G_MOUSE ACDSB_MOUSE ACADS_MOUSE SODC_MOUSE TCTP_MOUSE TPP1_MOUSE MYG1_MOUSE 502 711* 927* 973* 811* 707 181 166* 172* 868 469* 1047* 85 104 779* 911 177* 473 785* 222 466 511 1030* 1029* 948 820* 1050* 564* 13200 55949 276770 16852 14667 26384 15519 15163 15163 229279 98758 56357 74142 74142 68349 56530 319518 14569 11857 232201 107702 20715 66885 11409 20655 22070 12751 60315 246221 15510 11464 11746 13039 114584 72042 109672 78920 Gene entrez ID 3-mercaptopyruvate sulfurtransferase 60 kDa heat shock protein, mitochondrial Actin, alpha cardiac muscle Annexin A4 Cathepsin L1 Chloride intracellular channel protein Coactosin-like protein Cytochrome b5 Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial Dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48kDa subunit Elongation factor 1-beta Eukaryotic translation initiation factor 5A-1 Galectin-1 Ganglioside GM2 activator Glucosamine-6-phosphate isomerase Heat shock protein HSP 90-alpha Hematopoietic lineage cell-specific protein Hematopoietic lineage cell-specific protein Heterogeneous nuclear ribonucleoprotein A3 Heterogeneous nuclear ribonucleoprotein F Isovaleryl-CoA dehydrogenase, mitochondrial Lon protease homolog, mitochondrial Lon protease homolog, mitochondrial NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial Protein canopy homolog Pyruvate dehydrogenase phosphatase regulatory subunit, mitochondrial Rab GDP dissociation inhibitor beta Rho GDP-dissociation inhibitor Rho GTPase-activating protein 25 Ribonuclease inhibitor Serine protease inhibitor A3G Short/branched chain specific acyl-CoA dehydrogenase, mitochondrial Short-chain specific acyl-CoA dehydrogenase, mitochondrial Superoxide dismutase [Cu-Zn] Translationally-controlled tumor protein Tripeptidyl-peptidase UPF0160 protein MYG1 Protein Name -1.70 -1.76 -2.08 1.82 -1.65 -4.50 -3.30 -1.62 -1.76 1.52 -1.51 6.23 -1.55 -2.18 2.08 3.90 -1.58 2.01 -2.70 Fold change (‘C57BL/6' /'BALB/c') -6.15 -1.84 -1.61 -3.36 -1.54 1.51 2.80 1.78 -2.10 -2.21 -2.37 1.52 -1.97 -4.49 -3.85 -2.03 2.15 1.60 1.70 -1.53 1.60 4.80 -1.62 -2.16 -2.21 -2.33 2.49 3.24 -1.73 -1.61 2.32 -3.44 Fold change (‘C57BL/6+IFN' /'BALB/c+IFN') -1.70 -1.85 -1.66 -1.98 -1.68 1.61 4.99 49.0 24.7 16.8 14.9 20.8 32.5 84.7 54.2 54.2 39.6 45.7 46.3 105.8 105.8 30.1 20.8 99.2 50.5 22.8 73.3 49.8 49.0 47.8 44.9 15.9 19.4 61.3 42.7 33.0 60.9 42.0 36.0 37.5 27.0 15.9 15.2 49.0 MW (kDa) 5.52 4.53 5.08 5.32 5.63 4.93 4.73 4.73 9.1 5.31 8.53 6.15 6.15 6.67 4.95 5.93 5.93 4.97 6.03 4.69 6.06 8.96 6.02 4.76 6.1 6.54 6.11 5.91 5.23 5.43 6.37 5.09 5.28 4.96 9.11 Protein pI Dissertation A15 Each of 35 proteins had all of identified isoforms existed in higher levels in BMMs of one mouse strain Symbol * indicate protein spots which identified at different levels in BMM-BALB/c and BMM-C57BL/6 in both control and IFN-γ treatment condition Positive fold change value means higher in BMM-C57BL/6, negative value means lower in BMM-C57BL/6 THTM_MOUSE CH60_MOUSE ACTC_MOUSE ANXA4_MOUSE CATL1_MOUSE CLIC1_MOUSE COTL1_MOUSE CYB5_MOUSE ODO2_MOUSE Accession Number 716 524* 959* 664 647 732* 960 938* 449* Spot Table A.5: Thirty five proteins of which total amount was significant different in two strains Supplements Supplements C57BL/6 vs BALB/c 2.76 -1.63 1.80 -1.57 -2.12 -1.75 -2.04 -1.50 -1.51 -1.71 2.01 -1.52 -1.56 -1.81 -1.79 -1.52 2.04 -1.99 -1.63 2.86 C57BL/6+IFN vs BALB/c+IFN 3.19 2.23 -2.16 1.68 2.29 -1.55 -1.68 -1.89 -1.64 -1.68 -2.21 1.56 1.65 1.98 2.49 -1.56 1.95 2.32 -1.52 1.60 2.32 -1.57 2.03 1.73 -1.68 -1.78 2.30 -1.67 -2.01 -1.64 -1.82 1.56 -1.66 -1.50 2.32 Spot 182* 184* 187 191 192 197* 198 204 205* 213 216* 217 265 270 281 288* 289 311* 319* 323 332 344 346* 377* 385* 400* 417* 443* 444* 472* 481 486 491 494* 509* 538* 541 550 553 554* 579 C57BL/6 vs BALB/c -1.78 -4.46 1.60 -1.75 -2.50 -1.49 -1.92 1.99 -1.78 1.59 -1.55 -1.81 -1.84 -1.56 -1.54 -1.59 -1.67 -1.83 -2.24 2.04 1.75 -1.60 -1.62 -1.70 -1.67 -1.78 1.67 -1.64 - C57BL/6+IFN vs BALB/c+IFN -1.76 -2.89 1.56 -1.84 1.60 1.59 -1.51 1.53 -2.14 -1.99 -1.53 1.63 -1.82 -1.99 -3.01 -1.78 -1.55 -3.38 -1.87 -3.14 -3.28 2.27 1.78 -1.86 -3.24 -1.64 -2.10 -2.13 -1.97 1.58 -1.54 -1.72 -1.76 Spot 581* 600* 601 653* 668 669 670* 671* 679* 685 690 695* 703* 713 715 748 755 761* 764 768* 771 774 781* 786* 807* 852* 864* 917 926 931* 962* 964 982* 983 988* 990 992* 996* 1002 1003* 1004 C57BL/6 vs BALB/c -1.98 -2.26 -2.36 -1.85 -13.41 2.00 -1.61 -2.19 1.82 1.54 -1.78 -1.59 -1.50 2.34 2.49 -1.97 -1.59 2.54 2.34 4.11 1.70 -1.59 -2.71 -1.55 -1.58 -2.90 1.51 -2.42 -1.64 -1.56 - C57BL/6+IFN vs BALB/c+IFN -2.83 -1.74 -1.75 -2.46 -2.95 -2.13 1.72 -1.54 1.50 2.27 1.91 2.07 -1.78 -1.68 -1.58 2.60 -1.72 -1.65 -1.58 1.82 1.73 3.15 -2.29 -1.64 -3.99 -1.59 1.98 -3.19 -3.02 -3.07 -1.57 -2.10 -1.54 Spot 1009* 1013 1018* 1023* 1024* 1025* 1031 1042 1043 1046 1052* 1060* 1065 C57BL/6 vs BALB/c -1.86 -2.02 -1.54 4.22 5.09 -1.57 1.50 1.92 - Dissertation C57BL/6+IFN vs BALB/c+IFN -2.22 -1.96 4.27 4.85 -1.85 -3.17 1.52 1.62 -2.25 1.61 2.32 -1.50 Table A.6: Fold change of 136 protein spots being present at different levels in a strain dependent manner which were not identified by MALDI-MS/MS Spot 1* 5* 14* 19 26 28 32* 44 46* 60 62 65* 66* 69 71 73 74* 83* 89 90 92 94 96 99* 101 106 107 108 119 121* 125* 134 139* 151 157 160 164 168 174 175* A16 Dissertation IPI00330480 IPI00153381* IPI00114945* IPI00224626* IPI00453792 IPI00136293* IPI00857151* IPI00135646* IPI00314069* IPI00857226* IPI00131701 IPI00469942 IPI00380436* IPI00118899* IPI00125653 IPI00230440* IPI00553798* IPI00169980* IPI00116339* IPI00466128 IPI00223757 IPI00129350* IPI00330482 IPI00405699* IPI00856379 IPI00468203* IPI00877279 IPI00877291 IPI00317309* IPI00121576 IPI00625950 IPI00353418 IPI00322312* IPI00122568 IPI00133218 Protein Gene Entrez ID 432987 66152 18000 235072 319518 67759 217830 19299 56360 14081 50790 68738 109711 60595 11520 269378 66395 107197 11629 58810 11677 56454 67689 212647 11674 12306 11745 11746 11747 68316 11821 232201 192662 11857 67166 1110020P15Rik 40422.0 40428.0 4930402E16Rik 5033414D02Rik 9030617O03Rik Abcd3 Acot9 Acsl1 Acsl4 Acss1 Actn1 Actn4 Adfp Ahcy Ahnak AI462493 Aif1 Akr1a4 Akr1b3 Aldh18a1 Aldh3b1 Aldh4a1 Aldoa Anxa2 Anxa3 Anxa4 Anxa5 Apoo Aprt Arhgap25 Arhgdia Arhgdib Arl8b Gene Symbol A17 35 kDa protein Cytochrome b-c1 complex subunit Septin-2 Septin-7 Pyruvate dehydrogenase phosphatase regulatory subunit, mitochondrial Transmembrane protein C9orf46 homolog Isoform of UPF0317 protein C14orf159 homolog, mitochondrial ATP-binding cassette, sub-family D, member Acyl-coenzyme A thioesterase 9, mitochondrial Long-chain-fatty-acid CoA ligase Isoform Long of Long-chain-fatty-acid CoA ligase Acetyl-coenzyme A synthetase 2-like, mitochondrial Alpha-actinin-1 Alpha-actinin-4 adipose differentiation related protein Adenosylhomocysteinase AHNAK nucleoprotein isoform UPF0723 protein C11orf83 homolog Allograft inflammatory factor Alcohol dehydrogenase [NADP+] Aldose reductase Isoform Short of Delta-1-pyrroline-5-carboxylate synthetase Aldehyde dehydrogenase family member B1 Delta-1-pyrroline-5-carboxylate dehydrogenase, mitochondrial Fructose-bisphosphate aldolase Annexin A2 Annexin A3 annexin A4 Annexin A5 Apolipoprotein O adenine phosphoribosyl transferase Isoform of Rho GTPase-activating protein 25 Rho GDP-dissociation inhibitor Rho GDP-dissociation inhibitor ADP-ribosylation factor-like protein 8B Protein name Fold change (C57BL/6' /'BALB/c') 1.52 -1.71 -1.79 2.46 1.70 1.95 1.60 1.73 -1.78 -1.58 -1.67 -1.70 -1.82 3.66 -6.21 1.87 2.32 -1.55 -2.26 1.58 -1.95 - Fold change (C57BL/6+IFN' /'BALB/c+IFN') 2.67 1.66 -2.04 -1.82 1.71 2.38 1.90 1.50 1.68 1.98 1.78 -1.78 -1.73 1.54 -1.67 -1.71 2.90 -3.63 -1.50 -2.17 1.75 1.52 2.37 -1.93 -1.60 -1.75 -1.60 -2.59 -1.96 -1.50 -2.10 -2.08 1.52 Table A.7: A total of 343 proteins were identified at different levels in BMM-BALB/c and BMM-C57BL/6 in control and/or IFN-γ treatment condition by LC-MS/MS approach Supplements Supplements Protein IPI00125266* IPI00134746 IPI00330303 IPI00130280* IPI00468481* IPI00341282* IPI00133342* IPI00407692 IPI00118787 IPI00120684 IPI00230422 IPI00122442 IPI00415385 IPI00330627* IPI00399958 IPI00406790 IPI00137331 IPI00277930 IPI00308938* IPI00757359 IPI00125676 IPI00320217 IPI00116283* IPI00331174 IPI00469268* IPI00112787 IPI00117031* IPI00113849 IPI00890117* IPI00157508* IPI00223047 IPI00136703* IPI00130344* IPI00153903* IPI00315879 IPI00222125 IPI00323600* IPI00124820 IPI00135390* IPI00117978* IPI00120719 Gene Entrez ID 11886 11898 108147 11946 11947 11950 27425 11964 73834 12028 27061 12036 72567 109778 12321 108058 12331 12332 12334 53872 12362 12461 12462 12468 12469 57781 12514 12540 12631 12655 216197 12709 114584 69634 66054 69156 12721 23790 78885 12857 12858 Gene Symbol Asah1 Ass1 Atic Atp5a1 Atp5b Atp5f1 Atp5l Atp6v1a Atp6v1d Bax Bcap31 Bcat2 Bclaf1 Blvra Calu Camk2d Cap1 Capg Capn2 Caprin1 Casp1 Cct2 Cct3 Cct7 Cct8 Cd200r1 Cd68 Cdc42 Cfl1 Chi3l3 Ckap4 Ckb Clic1 Clybl Cndp2 Comtd1 Coro1a Coro1c Coro7 Cox4i1 Cox5a Protein name Acid ceramidase Argininosuccinate synthase 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase ATP synthase subunit alpha, mitochondrial ATP synthase subunit beta, mitochondrial ATP synthase subunit b, mitochondrial ATP synthase subunit g, mitochondrial Isoform of V-type proton ATPase catalytic subunit A V-type proton ATPase subunit D Apoptosis regulator BAX B-cell receptor-associated protein 31 Branched-chain-amino-acid aminotransferase, mitochondrial Isoform of Bcl-2-associated transcription factor Biliverdin reductase A calumenin isoform Isoform of Calcium/calmodulin-dependent protein kinase type II delta chain Adenylyl cyclase-associated protein Macrophage-capping protein Calpain-2 catalytic subunit cytoplasmic activation/proliferation-associated protein isoform c Caspase-1 T-complex protein subunit beta T-complex protein subunit gamma T-complex protein subunit eta T-complex protein subunit theta Cell surface glycoprotein CD200 receptor Isoform Short of Macrosialin Isoform of Cell division control protein 42 homolog Cofilin-1 Chitinase-3-like protein Cytoskeleton-associated protein Creatine kinase B-type Chloride intracellular channel protein Citrate lyase subunit beta-like protein, mitochondrial Cytosolic non-specific dipeptidase Catechol-O-methyltransferase domain-containing protein Coronin-1A Coronin-1C Coronin-7 Cytochrome c oxidase subunit isoform 1, mitochondrial Cytochrome c oxidase subunit 5A, mitochondrial A18 Fold change (C57BL/6' /'BALB/c') 1.83 1.65 1.56 1.56 1.58 2.00 -1.73 -1.80 -1.68 -1.56 -1.55 -1.67 2.16 1.56 -1.83 12.99 1.56 -1.90 -1.96 2.34 -1.65 -1.78 -2.00 1.69 1.63 Dissertation Fold change (C57BL/6+IFN' /'BALB/c+IFN') 2.02 -2.00 1.62 1.65 1.62 1.78 1.52 -1.62 2.42 1.56 2.70 -2.06 -2.20 -1.90 -1.65 -1.76 -2.09 -2.13 -2.65 -1.67 -1.83 -1.59 -1.67 1.79 -2.05 13.89 -2.55 -2.33 2.90 -1.60 1.59 -2.57 -2.63 1.50 - 235339 13430 12934 56431 13424 93747 55949 66656 13629 27984 233637 78294 621837 667618 98878 13063 54127 13822 IPI00116154* IPI00225390 IPI00114377 IPI00330094 IPI00881401 IPI00125931 IPI00658539 IPI00404551 IPI00118987* IPI00128154* IPI00309520* IPI00230113* IPI00315794* IPI00119131* IPI00121079* IPI00117117* IPI00330476 IPI00230035 IPI00420363 IPI00387379* IPI00134506* IPI00331549 IPI00153660 IPI00131445 IPI00114375 IPI00127942 IPI00119876 IPI00454049* IPI00320208* IPI00118875* IPI00466069 IPI00226872 IPI00762705 IPI00470152 IPI00622837 IPI00626366* IPI00318671* IPI00265239* IPI00137736* IPI00330289* Protein Gene Entrez ID 12859 110323 12866 12894 12896 13014 19025 13033 13036 13039 13040 109672 66427 72017 109754 13058 20430 13205 13207 67460 27369 52585 Supplements Dlat Dnm2 Dpysl2 Dstn Dync1h1 Echs1 Eef1b2 Eef1d Eef2 Efhd2 EG233637 Rps27a EG621837 EG667618 Ehd4 Cycs Rps28 Epb4.1l2 Cox5b Cox6b1 Cox7a2 Cpt1a Cpt2 Cstb Ctsa Ctsd Ctsh Ctsl Ctss Cyb5 Cyb5b Cyb5r1 Cyb5r3 Cybb Cyfip1 Ddx3x Ddx5 Decr1 Dguok Dhrs1 Gene Symbol A19 Cytochrome c oxidase subunit 5B, mitochondrial Cytochrome c oxidase subunit VIb isoform Cytochrome c oxidase polypeptide 7A2, mitochondrial Carnitine O-palmitoyltransferase 1, liver isoform Carnitine O-palmitoyltransferase 2, mitochondrial Cystatin-B cathepsin A isoform a Cathepsin D Cathepsin H Cathepsin L1 Cathepsin S 11 kDa protein Cytochrome b5 type B Isoform of NADH-cytochrome b5 reductase Isoform of NADH-cytochrome b5 reductase Cytochrome b-245 heavy chain Isoform of Cytoplasmic FMR1-interacting protein ATP-dependent RNA helicase DDX3X Probable ATP-dependent RNA helicase DDX5 2,4-dienoyl-CoA reductase, mitochondrial Isoform of Deoxyguanosine kinase, mitochondrial Dehydrogenase/reductase SDR family member Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Isoform of Dynamin-2 Dihydropyrimidinase-related protein Destrin Cytoplasmic dynein heavy chain Enoyl-CoA hydratase, mitochondrial Elongation factor 1-beta eukaryotic translation elongation factor delta isoform b Elongation factor EF-hand domain-containing protein D2 similar to Twinfilin, actin-binding protein, homolog ribosomal protein S27a hypothetical protein similar to Acidic ribosomal phosphoprotein P0 EH-domain containing 4-KJR (Fragment) Cytochrome c, somatic 40S ribosomal protein S28 Band 4.1-like protein Protein name 1.79 -1.95 -1.76 -1.59 -1.85 -1.74 1.59 -2.05 -1.54 Fold change (C57BL/6' /'BALB/c') 1.80 -1.52 4.37 -2.27 1.66 1.66 1.96 2.18 1.96 2.89 -1.50 1.68 -2.73 1.51 -1.64 -1.69 -1.82 -1.64 1.69 -2.66 -2.38 -1.58 -2.67 -1.97 1.74 -1.98 -1.91 1.69 -2.85 -1.71 Fold change (C57BL/6+IFN' /'BALB/c+IFN') 1.59 2.36 1.51 1.53 1.76 2.04 1.56 3.34 -1.79 1.69 1.63 2.82 1.87 1.63 2.19 -1.80 -1.55 1.72 -1.87 1.61 Dissertation Supplements Protein IPI00121440 IPI00753434* IPI00330862 IPI00116705* IPI00330695 IPI00122015* IPI00169731* IPI00921658* IPI00117181* IPI00673288* IPI00117063 IPI00228385* IPI00310090 IPI00112129* IPI00124675 IPI00323251 IPI00230283* IPI00123814 IPI00464317* IPI00169870* IPI00119095* IPI00347770* IPI00775863 IPI00317740* IPI00221426 IPI00877205 IPI00228633 IPI00890906* IPI00119058* IPI00117083 IPI00117167* IPI00172039 IPI00378480 IPI00110805* IPI00121908* IPI00113299* IPI00114492* IPI00850057* IPI00624138* IPI00109996* IPI00222809* Gene Entrez ID 110826 100047333 22350 11770 227737 223601 108101 192176 14251 634386 233908 14381 50917 67092 14468 14469 28030 11605 14660 234407 14667 242851 14693 14694 75612 14718 14751 93695 14784 17713 227753 14960 26914 14964 14999 14969 14972 100044874 14964 14980 100198 Gene Symbol Etfb Evl Ezr Fabp4 Fam129b Fam49b Fermt3 Flna Flot1 Ftl1 Fus G6pdx Galns Gatm Gbp1 Gbp2 Gfm1 Gla Gls Glt25d1 Gm2a Gnat3 Gnb2 Gnb2l1 Gns Got1 Gpi1 Gpnmb Grb2 Grpel1 Gsn H2-Aa H2afy H2-D1 H2-DMb1 H2-Eb1 H2-K1 H2-K1 H2-L H2-L H6pd Protein name Electron transfer flavoprotein subunit beta Isoform of Ena/VASP-like protein Ezrin Fatty acid-binding protein, adipocyte Niban-like protein Protein FAM49B Fermitin family homolog Isoform of Filamin-A Flotillin-1 ferritin light chain 53 kDa protein Glucose-6-phosphate 1-dehydrogenase X N-acetylgalactosamine-6-sulfatase Glycine amidinotransferase, mitochondrial Interferon-induced guanylate-binding protein Interferon-induced guanylate-binding protein Elongation factor G 1, mitochondrial alpha-galactosidase A glutaminase isoform Glycosyltransferase 25 family member Ganglioside GM2 activator Guanine nucleotide-binding protein G(t) subunit alpha-3 Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 Guanine nucleotide-binding protein subunit beta-2-like N-acetylglucosamine-6-sulfatase Aspartate aminotransferase, cytoplasmic Glucose-6-phosphate isomerase glycoprotein (transmembrane) nmb Isoform of Growth factor receptor-bound protein GrpE protein homolog 1, mitochondrial Isoform of Gelsolin H-2 class II histocompatibility antigen, I-E alpha chain (Fragment) Isoform of Core histone macro-H2A.1 H-2 class I histocompatibility antigen, D-D alpha chain Class II histocompatibility antigen, M beta chain H-2 class II histocompatibility antigen, I-A beta chain mRNA H-2 class I histocompatibility antigen, K-D alpha chain H-2D cell surface glycoprotein (Fragment) H-2 class I histocompatibility antigen, L-D alpha chain Hexose-6-phosphate dehydrogenase A20 Fold change (C57BL/6' /'BALB/c') 1.56 -1.81 1.75 -1.60 -1.58 -1.64 2.58 1.73 -2.66 -1.54 -2.55 3.36 1.99 1.65 1.97 2.12 -1.63 8.13 -1.71 -1.69 -2.23 1.51 -2.97 -2.84 4.78 34.48 -3.81 3.79 -24.33 3.34 Dissertation Fold change (C57BL/6+IFN' /'BALB/c+IFN') -2.50 -1.55 2.52 -1.60 -1.90 -1.77 -1.76 2.00 1.72 -1.93 1.57 -4.28 -24.01 2.77 2.32 1.75 1.81 1.73 2.50 2.07 1.68 -2.09 1.74 -2.51 -1.70 6.25 -2.41 1.64 -1.57 -1.56 -2.33 15.63 500.00 -3.90 3.86 -46.91 4.22 IPI00762198* IPI00115530* IPI00317488* IPI00284806 IPI00626132* IPI00110224* IPI00331628* IPI00330804* IPI00229080* IPI00320165 IPI00453499* IPI00123570 IPI00136858* IPI00467447* IPI00111285 IPI00757372* IPI00761784 IPI00471246* IPI00421206 IPI00462934 IPI00798492 IPI00622240 IPI00466711* IPI00125091* IPI00229517* IPI00130627 IPI00129265 IPI00462072* IPI00230507 IPI00139795* IPI00849349* IPI00319188 IPI00119063 IPI00469307 IPI00169818 IPI00107952* IPI00118011* IPI00117896* IPI00229534* IPI00128450* IPI00222546 Protein Supplements Gene Entrez ID 15129 15212 52120 15452 15108 114664 15488 15519 15516 53415 381314 15953 16181 29875 16365 664994 16409 56357 239217 16549 16661 16681 16784 16796 16852 19141 16889 13806 71679 67186 100048461 16956 16971 16976 211228 17105 110173 13589 17118 23943 19934 Hbb-b1 Hexb Hgsnat Hprt1 Hsd17b10 Hsd17b11 Hsd17b4 Hsp90aa1 Hsp90ab1 Htatip2 Iars2 Ifi47 Il1rn Iqgap1 Irg1 Isoc2a Itgam Ivd Kctd12 Khsrp Krt10 Krt2 Lamp2 Lasp1 Lgals1 Lgmn Lipa Eno1 LOC100044492 LOC100048062 LOC100048461 Lpl Lrp1 Lrpap1 Lrrc25 Lyz2 Manba Mapre1 Marcks Mbc2 Rpl22 Gene Symbol A21 Beta-globin Beta-hexosaminidase subunit beta Heparan-alpha-glucosaminide N-acetyltransferase Hypoxanthine-guanine phosphoribosyltransferase Hydroxysteroid (17-beta) dehydrogenase 10 Isoform of Estradiol 17-beta-dehydrogenase 11 Peroxisomal multifunctional enzyme type Heat shock protein HSP 90-alpha MCG18238 Oxidoreductase HTATIP2 Isoleucyl-tRNA synthetase, mitochondrial Interferon gamma inducible protein 47 Isoform of Interleukin-1 receptor antagonist protein Ras GTPase-activating-like protein IQGAP1 Immune-responsive gene protein Isochorismatase domain-containing protein 2A, mitochondrial Integrin alpha-M Isovaleryl-CoA dehydrogenase, mitochondrial BTB/POZ domain-containing protein KCTD12 Far upstream element-binding protein keratin complex 1, acidic, gene 10 71 kDa protein Lysosomal membrane glycoprotein 2, isoform CRA_a LIM and SH3 protein Galectin-1 Legumain lysosomal acid lipase A Alpha-enolase hypothetical protein similar to LOC665931 protein isoform similar to dendritic cell-associated C-type lectin-1; DECTIN-1 lipoprotein lipase Prolow-density lipoprotein receptor-related protein Alpha-2-macroglobulin receptor-associated protein Leucine-rich repeat-containing protein 25 Lysozyme C-2 Beta-mannosidase Microtubule-associated protein RP/EB family member Myristoylated alanine-rich C-kinase substrate Isoform of Extended synaptotagmin-1 60S ribosomal protein L22 Protein name Fold change (C57BL/6' /'BALB/c') 5.65 1.66 4.93 1.93 1.63 1.73 -1.67 -1.59 1.79 -1.58 -1.70 2.15 1.61 2.08 6.62 1.61 -1.72 -2.19 -1.60 -1.53 1.57 -1.77 5.75 1.54 1.59 2.23 2.87 -1.79 -2.00 1.94 - Fold change (C57BL/6+IFN' /'BALB/c+IFN') 5.21 1.81 4.39 -1.83 1.82 1.58 1.64 -1.84 -1.74 1.74 1.69 -1.90 -1.89 -1.76 1.79 1.93 2.05 -1.64 -1.68 3.00 1.58 -2.41 -2.15 1.62 -1.89 -1.72 6.06 1.68 1.83 2.88 2.56 -2.48 -3.02 1.89 -1.78 Dissertation Supplements Protein IPI00849952 IPI00411071* IPI00132874 IPI00137706* IPI00604945 IPI00118963* IPI00754363* IPI00222514 IPI00750596* IPI00355248* IPI00131176 IPI00480233* IPI00380896* IPI00123181 IPI00109044 IPI00118120* IPI00320157* IPI00111831* IPI00315593 IPI00314726* IPI00124203* IPI00125960* IPI00116748* IPI00230715 IPI00121288* IPI00308882* IPI00229008 IPI00170093 IPI00228567* IPI00132444* IPI00132600 IPI00129186 IPI00132314 IPI00321058* IPI00626237* IPI00475158* IPI00458105 IPI00453607* IPI00119305* IPI00331552 IPI00128904 Gene Entrez ID 269878 17304 23945 17524 246221 56282 64656 218506 56428 17708 17709 17721 17879 17886 67268 17918 67111 17938 17939 27419 17972 17988 67273 67184 68342 227197 17993 225887 212114 66536 18145 67963 18220 246730 18293 624814 433745 18733 18813 18458 23983 Gene Symbol Megf8 Mfge8 Mgll Mpp1 Mpst Mrpl12 Mrps23 Mrps27 Mtch2 mt-Co1 mt-Co2 mt-Nd6 Myh1 Myh9 Mylc2b Myo5a Naaa Naca Naga Naglu Ncf4 Ndrg1 Ndufa10 Ndufa13 Ndufb10 Ndufs1 Ndufs4 Ndufs8 Nhlrc3 Nipsnap3a Npc1 Npc2 Nucb1 Oas1a Ogdh OTTMUSG00000005734 OTTMUSG00000008543 Pira11 Pa2g4 Pabpc1 Pcbp1 Protein name similar to Multiple epidermal growth factor-like domains Isoform of Lactadherin Monoglyceride lipase 55 kDa erythrocyte membrane protein 3-mercaptopyruvate sulfurtransferase 39S ribosomal protein L12, mitochondrial Mrps23 protein (Fragment) 28S ribosomal protein S27, mitochondrial Mitochondrial carrier homolog cytochrome c oxidase subunit I Cytochrome c oxidase subunit NADH dehydrogenase subunit Myosin-1 Myosin-9 Myosin regulatory light chain MRLC2 Myosin-Va N-acylethanolamine-hydrolyzing acid amidase Nascent polypeptide-associated complex subunit alpha Alpha-N-acetylgalactosaminidase Alpha-N-acetylglucosaminidase Neutrophil cytosol factor Protein NDRG1 NADH dehydrogenase [ubiquinone] alpha subcomplex subunit 10, mitochondrial NADH dehydrogenase [ubiquinone] alpha subcomplex subunit 13 NADH dehydrogenase [ubiquinone] beta subcomplex subunit 10 NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial NADH dehydrogenase [ubiquinone] iron-sulfur protein 4, mitochondrial NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, mitochondrial NHL repeat-containing protein Protein NipSnap homolog 3A Niemann Pick type C1 Epididymal secretory protein E1 Nucleobindin-1 2'-5'-oligoadenylate synthetase 1A Isoform of 2-oxoglutarate dehydrogenase E1 component, mitochondrial similar to NADH dehydrogenase (ubiquinone) Fe-S protein similar to ribosomal protein L3 Paired-Ig-like receptor A11 Proliferation-associated protein 2G4 Polyadenylate-binding protein Poly(rC)-binding protein A22 Fold change (C57BL/6' /'BALB/c') 3.02 -1.52 1.58 2.15 1.60 2.16 1.68 1.75 -2.29 2.55 1.58 1.51 1.87 2.26 -1.89 1.53 -2.52 -5.83 -1.70 -1.57 -2.60 1.70 1.56 2.08 -2.37 1.73 -1.89 -28.88 -1.57 -1.56 - Dissertation Fold change (C57BL/6+IFN' /'BALB/c+IFN') 1.63 2.93 1.58 -1.50 -1.94 1.53 1.80 1.68 1.56 -2.32 1.93 1.86 2.29 -2.74 1.51 1.61 -3.09 -6.74 -1.85 -2.20 -1.98 -2.62 -1.88 -2.24 1.55 1.67 1.74 1.51 -1.92 1.87 -1.91 2.39 -100.58 -1.65 -1.76 IPI00127707 IPI00918862* IPI00923036 IPI00222767 IPI00222496* IPI00283511* IPI00121013 IPI00224740* IPI00457898 IPI00407130 IPI00403031* IPI00776023 IPI00274656 IPI00315452 IPI00310567* IPI00400017 IPI00554989* IPI00132020 IPI00116192* IPI00915044* IPI00473190* IPI00654420 IPI00321190* IPI00124223* IPI00225419 IPI00153702 IPI00123278* IPI00653254 IPI00459279* IPI00116688 IPI00411115 IPI00137618 IPI00134621 IPI00110910* IPI00113503* IPI00225634* IPI00134599 IPI00136984 IPI00850840* IPI00134131 IPI00338536 Protein Supplements Gene Entrez ID 18521 66904 18571 27402 71853 216134 18611 18643 18648 18746 102595 102866 73078 18950 330260 19024 268373 72461 11757 54683 19124 114863 19156 19186 15170 217057 69051 110095 110391 19340 235442 19354 19384 14961 19893 20042 27050 20115 16785 20280 67680 Pcbp2 Pccb Pdcd6ip Pdhx Pdia6 Pdxk Pea15a Pfn1 Pgam1 Pkm2 Plekho2 Pls3 Pmpcb Pnp1 Pon2 Ppfibp2 Ppia Prcp Prdx3 Prdx5 Procr Prosc Psap Psme1 Ptpn6 Ptrh2 Pycr2 Pygl Qdpr Rab3d Rab8b Rac2 Ran H2-Ab1 Rpgr Rps12 Rps3 Rps7 Rpsa Scp2 Sdhb Gene Symbol A23 Isoform of Poly(rC)-binding protein Propionyl-CoA carboxylase beta chain, mitochondrial Isoform of Programmed cell death 6-interacting protein Pyruvate dehydrogenase protein X component, mitochondrial Protein disulfide-isomerase A6 Pyridoxal kinase Isoform of Astrocytic phosphoprotein PEA-15 Profilin-1 Phosphoglycerate mutase Isoform M2 of Pyruvate kinase isozymes M1/M2 Pleckstrin homology domain-containing family O member Plastin-3 Mitochondrial-processing peptidase subunit beta Purine nucleoside phosphorylase Serum paraoxonase/arylesterase Isoform of Liprin-beta-2 Peptidyl-prolyl cis-trans isomerase Lysosomal Pro-X carboxypeptidase Thioredoxin-dependent peroxide reductase, mitochondrial Isoform Mitochondrial of Peroxiredoxin-5, mitochondrial Protein C receptor, endothelial, isoform CRA_b Proline synthetase co-transcribed bacterial homolog protein Sulfated glycoprotein Proteasome activator complex subunit Isoform of Tyrosine-protein phosphatase non-receptor type Peptidyl-tRNA hydrolase 2, mitochondrial Pyrroline-5-carboxylate reductase Phosphorylase Dihydropteridine reductase Ras-related protein Rab-3D Ras-related protein Rab-8B Ras-related C3 botulinum toxin substrate GTP-binding nuclear protein Ran H-2 class II histocompatibility antigen, A-D beta chain Retinitis pigmentosa GTPase regulator (Fragment) 40S ribosomal protein S12 40S ribosomal protein S3 40S ribosomal protein S7 40S ribosomal protein SA Isoform SCP2 of Non-specific lipid-transfer protein Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial Protein name Fold change (C57BL/6' /'BALB/c') 1.60 3.08 -1.80 -1.50 -1.79 2.24 -1.56 1.73 1.68 2.38 1.93 -1.57 1.81 2.00 -1.78 1.91 -3.57 1.88 -1.77 -1.58 1.57 - Fold change (C57BL/6+IFN' /'BALB/c+IFN') -1.98 1.69 -1.73 1.59 2.89 -1.80 -2.11 -1.59 -1.88 -1.66 -2.31 -1.85 1.53 -1.77 1.97 1.76 -1.78 1.64 1.69 1.59 2.95 -1.63 2.36 -1.66 -1.98 1.96 -1.88 -2.24 1.61 -1.82 -1.85 -4.00 1.56 -2.04 -1.69 -1.90 -2.54 -1.77 Dissertation Supplements Protein IPI00454142 IPI00669817 IPI00121471 IPI00119079* IPI00115454* IPI00318175 IPI00122265* IPI00875340* IPI00308162* IPI00131584 IPI00404341 IPI00776208 IPI00420136* IPI00112053 IPI00130589* IPI00109109* IPI00313998* IPI00318485 IPI00408243 IPI00110852* IPI00122346 IPI00467004* IPI00121514 IPI00115117 IPI00125778 IPI00620227* IPI00322543 IPI00459493* IPI00124700* IPI00126861* IPI00132958* IPI00465786 IPI00132768* IPI00120083* IPI00831626* IPI00421223* IPI00403810* IPI00338039 IPI00169463* IPI00117352* IPI00226993* Gene Entrez ID 52398 20715 20719 20706 14057 27029 56726 20514 78830 57279 56857 57319 54198 69178 20655 20656 59010 109552 381760 107513 20832 20846 20867 66592 21346 21356 21391 21454 22042 21817 66834 21894 66154 74122 22003 326618 22146 22151 227613 22154 22166 Gene Symbol Sept11 Serpina3g Serpinb6a Serpinb9b Sfxn1 Sgsh Sh3bgrl Slc1a5 Slc25a12 Slc25a20 Slc37a2 Smpdl3a Snx3 Snx5 Sod1 Sod2 Sqrdl Sri Ssbp1 Ssr1 Ssr4 Stat1 Stip1 Stoml2 Tagln2 Tapbp Tbxas1 Tcp1 Tfrc Tgm2 Them2 Tln1 Tmem14c Tmem43 Tpm1 Tpm4 Tuba1c Tubb2a Tubb2c Tubb5 Txn1 Protein name Isoform of Septin-11 Serine protease inhibitor A3G Serpin B6 Serine (Or cysteine) peptidase inhibitor, clade B, member 9b Sideroflexin-1 N-sulfoglucosamine sulfohydrolase SH3 domain-binding glutamic acid-rich-like protein 58 kDa protein Calcium-binding mitochondrial carrier protein Aralar1 Mitochondrial carnitine/acylcarnitine carrier protein Isoform of Sugar phosphate exchanger acid sphingomyelinase-like phosphodiesterase 3a Sorting nexin-3 Sorting nexin-5 Superoxide dismutase [Cu-Zn] Superoxide dismutase [Mn], mitochondrial Sulfide:quinone oxidoreductase, mitochondrial Isoform of Sorcin single-stranded DNA binding protein isoform Translocon-associated protein alpha, muscle specific isoform Signal sequence receptor, delta, isoform CRA_c Signal transducer and activator of transcription Stress-induced-phosphoprotein Stomatin-like protein Transgelin-2 Isoform Short of Tapasin Thromboxane-A synthase Isoform of T-complex protein subunit alpha B Transferrin receptor protein Protein-glutamine gamma-glutamyltransferase Thioesterase superfamily member Talin-1 Transmembrane protein 14C Transmembrane protein 43 29 kDa protein Tropomyosin alpha-4 chain Tubulin alpha-1C chain Tubulin beta-2A chain Tubulin beta-2C chain Tubulin beta-5 chain Thioredoxin A24 Fold change (C57BL/6' /'BALB/c') -2.44 1.50 -1.94 -1.99 1.68 -1.80 -1.54 1.82 1.58 1.54 1.96 1.54 -2.25 2.15 2.79 1.53 -1.65 -4.13 -2.22 1.90 1.61 1.55 -1.90 -1.82 -1.51 -1.59 -1.68 -1.50 Dissertation Fold change (C57BL/6+IFN' /'BALB/c+IFN') -2.53 -21.07 -2.27 -4.22 1.51 -2.08 -2.26 -2.24 1.79 2.54 -2.20 3.24 -1.78 -1.66 -1.84 1.82 1.52 -2.23 1.59 -2.19 -1.60 -1.82 2.64 -1.60 -5.45 -2.51 2.04 -1.69 1.77 1.58 -2.07 -2.20 -1.79 -2.38 -2.85 -2.37 -2.18 50493 227620 22201 22195 22192 394432 67003 22225 22330 22335 80743 30930 65114 67776 22388 54401 22628 22629 22630 22631 22793 IPI00462482 IPI00123313* IPI00116718 IPI00169448 IPI00417181* IPI00119138* IPI00113214 IPI00405227* IPI00122548 IPI00120923* IPI00329942 IPI00111181 IPI00221817 IPI00314748* IPI00230682* IPI00230707 IPI00227392* IPI00853924* IPI00116498 IPI00387422* Zyx Ywhaz Ywhaq Ywhah Ywhag Ywhab Wdr1 Vwa5a Vps35 Vps26a Vps16 Vdac3 Vcl Usp5 Uqcrc2 Ugt1a5 Ube2m Ube2l3 Uba1 Uap1l1 Txnrd1 Txndc12 Gene Symbol Zyx protein 14-3-3 protein zeta/delta Isoform of 14-3-3 protein theta 14-3-3 protein eta 14-3-3 protein gamma Isoform Long of 14-3-3 protein beta/alpha WD repeat-containing protein Loss of heterozygosity 11 chromosomal region gene A protein homolog Vacuolar protein sorting-associated protein 35 Isoform of Vacuolar protein sorting-associated protein 26A Isoform of Vacuolar protein sorting-associated protein 16 homolog Voltage-dependent anion-selective channel protein Vinculin Ubiquitin carboxyl-terminal hydrolase Cytochrome b-c1 complex subunit 2, mitochondrial UDP-glucuronosyltransferase 1-7C NEDD8-conjugating enzyme Ubc12 Ubiquitin-conjugating enzyme E2 L3 Ubiquitin-like modifier-activating enzyme Isoform of UDP-N-acetylhexosamine pyrophosphorylase-like protein Isoform of Thioredoxin reductase 1, cytoplasmic Thioredoxin domain-containing protein 12 Protein name -1.99 - -1.82 -1.52 - -1.61 -1.95 - - - -1.96 - -1.86 - 1.75 1.75 - - -1.51 - -1.69 - Fold change (C57BL/6'/'BALB/c') -3.67 -2.02 -2.17 -1.78 -1.88 -2.01 -2.09 -1.80 -1.82 -2.10 -2.32 2.19 -2.32 -2.19 1.68 1.72 -2.35 -1.50 -1.69 -1.58 -2.26 1.55 Fold change (C57BL/6+IFN' /'BALB/c+IFN') Dissertation A25 Symbol * indicate proteins which differently expressed between two mouse strains in both control and IFN-γ treatment condition Positive fold change value means higher in BMM-C57BL/6, negative value means lower in BMM-C57BL/6 Thresholds for considering differences as significant were a minimum 1.5 fold change, and p ≤ 0.01 66073 IPI00776252* Gene Entrez ID IPI00133110 Protein Supplements [...]... generated by the “respiratory burst” spontaneously reacts to form hydrogen peroxide (H2O2), hydroxyl radicals (·OH), and hypochlorous acid (HOCl) The toxic oxidants produced by the respiratory burst are also able to react with those produced by inducible nitric oxide synthase (iNOS), thereby forming a large number of different toxic species (e.g., peroxynitrite) to mediate cytotoxicity by a wide variety of. .. susceptibility to various bacterial [27] and viral infections [28] During the innate inflammatory response, IFN- γ is produced mainly by natural killer cells and subsets of T lymphocytes, including natural killer T cells and CD8+ T cells [29] IFN- γ primarily signals through the Jak-Stat pathway, a pathway used by over 50 cytokines, growth factors, and hormones to affect gene regulation After the binding of IFN- γ... immune responses They also participate in the activation of T and B lymphocytes through the secretion of many cytokines Figure 1: Typical appearance of macrophage The cell surface exhibits many membrane extensions The cytoplasm contains some dense granules corresponding to lysosomes The Golgi apparatus is well developed The nucleus presents its typical lobate shape and a rather thick layer of dense... monocytes, together with lymphocytes, hepatocytes, endothelial cells, and dermal fibroblasts, also secrete IL-8 [16] This cytokine stimulates the chemotaxis of both neutrophils and T-cells, and inhibits IFN- γ release by human NK cells in vitro [17] The expression of the IL8 gene in monocytes is regulated by known inflammatory agents, such as LPS, PGE2, IL-1, TNFγ and IFN- γ [18] 1.1.3 Other functions of. .. fusing with phagosomes to form secondary lysosomes Within the secondary lysosomes, ingested cellular, bacterial and non-cellular material can be seen in various stages of degradation and digestion Microtubules and microfilaments are prominent in macrophages and form a well-organized, three-dimensional cytokeleton which surrounds the nucleus and extends throughout the cytoplasm to the cell periphery [5]... reported to kill tumour cells when activated with cytokines and endotoxin [20] Macrophages phagocytose aged erythrocytes during their circulation through the spleen The mechanism whereby macrophages recognize senescent cells is unknown Senescent red cells are sequestered in the spleen and their destruction presumably occurs because of a subtle abnormality detected by splenic macrophages Once ingested by macrophages, ... analyzing IFN- γ effects, the other two batches (BMM5 and 6) were used for studying effects of S aureus engulfment in macrophages (see table 1) 17 Materials and Methods Dissertation Table 1: BMM batches used in the study BMMbatches BMM1 BMM2 BMM3 BMM4 BMMsamples IFN- γ treatment S aureus infection Transcriptomic analysis Proteomic analysis Purpose +/- - no yes 2-DE proteomic map +/- - yes yes 2-DE proteomic. .. each spot (% volume) was determined by dividing the intensity of the spot by the sum of the intensities of all spots in the corresponding gel (per gel normalization) and the relative intensity of the corresponding spot on the internal standard gel labeled with Cy2 The mean relative spot intensities of all replicates of the different samples were used to evaluate the changes between groups A protein... plate and the extraction was repeated with 40 µl of the same solution The supernatants were dried completely at 40°C for 220 min Peptides were dissolved in 2.0 µl of matrix solution (3.2 mg/ml α-cyano-4-hydroxy cinnamic acid in 0.5% TFA and 50% ACN) and 0,7 µl of peptide solution spotted on the MALDI-target directly Prior to the measurement in the MALDI-TOF/TOF instrument the samples were allowed to dry... in cell morphology, motility, division and intracellular organization and transport Most recently, the proteomic analysis of changes in macrophage microtubule cytoskeleton proteins during activation was reported Microtubule cytoskeleton proteins were extracted from RAW264.7 macrophages which were stimulated with IFN- γ and lipopolysaccharide (LPS) Applying a LC-MS/MS aaproach, the analysis identified