monitoring protein protein interaction in living cells by acceptor photobleaching

... cellular proteins led to ReAsH dependent aggregation of the tagged protein as wells as endogenous binding partners By sequestering protein complexes in the aggregates, activity is inhibited Finally, ... approach for increasing the biarsenical-tetracysteine contrast is to attach multiple tetracysteines to a single protein By fusing several tetracysteines locally to a single protein, it should ... YRECCPGCCMWR, was discovered capable of inducing protein aggregation upon biarsenical labeling in living cells or in vitro1 Aggregation is initiated by labeling with ReAsH or CHoXAsH, but not with...

Ngày tải lên: 13/11/2014, 10:46

... of cells examined Combination of protein a1 (%) s1 (ns) Cerulean-elongin B Elongin B-cerulean Cerulean-elongin B-citrine-elongin C Cerulean-elongin B-elongin C-citrine Elongin B-cerulean-elongin ... cerulean-linked elongin B and citrine-linked elongin C can be detected in the following donor -acceptor combinations in decreasing order: cerulean-elongin B and citrine-elongin C > cerulean-elongin B ... elongin C induced by binding of elongin B Increased FRET signals between pVHL-cerulean and citrine-elongin C by coexpression of elongin B suggest that a conformation change of elongin C induced by...

Ngày tải lên: 16/03/2014, 05:20

... points regarding the mobility and binding of GFP-GR inside the nucleus First, as pointed out in [14], the primary rate kinetics or singlebinding state (Eq 1) can satisfactorily describe the binding ... have increased over the years Early analyses characterized diffusion alone [7,16-18] More recently, investigators have studied the interaction of GFPtagged proteins with binding sites inside living ... show a possible way of obtaining unique values for diffusion coefficient and binding rate parameters of biomolecules inside living cells A possible procedure for obtaining unique values for molecular...

Ngày tải lên: 13/08/2014, 16:21

... for studying rafts is interrupting rafts by changing the amount of cholesterol in cells, which includes increasing its amount by adding in extra cholesterol and extracting it by drug treatments ... 91 5.2.3 Interaction between EGFR and PTB stimulated by EGF 92 5.2.4 PTB domain translocation induced by EGF stimulation 97 5.2.5 Inhibition of EGFR-PTB interaction by inhibiting phosphorylation ... possibility of monitoring protein behaviour in situ Genetic GFP tagging makes the proteins of interest visible by the help of fluorescence imaging tools, the development of which has improved in the...

Ngày tải lên: 10/09/2015, 09:25

... fluorescent proteins using fluorescence lifetime imaging microscopy (FLIM) is a technique for the observation of protein protein interactions [14,15] This method can be applied to interactions in loose ... direct observation of the interaction between GAPDH and PGK chimeric proteins linked to cerulean [16] or citrine [17], a FRET pair, in living cells FRET imaging of interaction between GAPDH and ... expressed GAPDH–citrine (chimeric protein with N-terminal GAPDH and C-terminal citrine) and citrine–GAPDH (chimeric protein with N-terminal citrine and C-terminal GAPDH) were localized in the cytoplasm...

Ngày tải lên: 16/02/2014, 09:20

... blotting in response to three main signal transduction pathways in dog thyrocytes in primary culture [thyrotropin/cAMP, EGF–HGF/MAPK (mitogen-activated protein kinase) and PMA/PKC (protein kinase ... corresponded to 5¢ deleted proteins (Fig 7A) To verify whether the cytokeratin interaction with p76RBE obtained in the two-hybrid was specific, we tested the protein interaction in vitro p76RBE was expressed ... further examined the cellular interaction of RhoB and p76RBE using wt and mutated Rho proteins The MYCtagged RhoB proteins were coexpressed in COS cells with HA-tagged p76RBE and then isolated by immunoprecipitation...

Ngày tải lên: 23/03/2014, 21:20

... neuroepithelial cells into astrocytes, by binding and hyper activating the promoter of glial fibrillary acidic protein (GFAP) In parallel, BMP signals lead to enhanced expression of ID proteins, which in turn ... mitogen-activated protein kinase signaling pathway and induction of neurite outgrowth in PC12 cells by bone morphogenetic protein- 2 J Biol Chem 274, 26503–26510 Levine AJ & Brivanlou AH (2006) GDF3, a BMP inhibitor, ... usually by binding to ligand dimers Cell membrane Predimerization of type I receptors, type I–type II receptors Dimeric secreted proteins such as noggin, chordin, follistatin and GDF3 bind and therby...

Ngày tải lên: 19/02/2014, 00:20

... before being subjected to FACS analysis After exclusion of dead cells by 7-aminoactinomycin D staining, analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, Franklin Lakes, ... (corresponding to amino acids 144–152 of the murine PrP) on a predicted surface loop of L1–VLP as a fusion protein, oligonucleotides encoding the epitope were inserted by inverse-touchdown PCR into ... effectively inhibited PrPSc de novo synthesis in living cells This strongly suggests a functionally relevant protective role of the induced Ab to PrP (Fig 6), indicating that even previously infected cells...

Ngày tải lên: 16/03/2014, 11:20

... Bodipy GTPcS binding assay (A) Specific increase in GTPcS binding to the Gi1 (r) protein and not the GsL (j) protein in the presence of membranes containing the truncated cannabinoid receptor ... G proteins in the absence of ligands The fluorescent GTPcS binding experiment showed the specificity of the cannabinoid receptor to Gai1 protein and not GasL protein The radioactive GTPcS binding ... plate After days of incubation, the cells were harvested by spinning in a centrifuge; · 105 cells from each clone were lysed using 1% SDS in a buffer containing protease inhibitors and DNase...

Ngày tải lên: 23/03/2014, 07:20

... CXCL10 induction in fibroblasts by IL-1β and interferons CXCL8 and CXCL10 induction in fibroblasts by IL-1β and interferons Confluent fibroblast monolayers were incubated with IL-1β in combination ... No Proost et al Figure CXCL10 induction by combinations interferons in in fibroblasts human microvascular endothelial cells CXCL10 induction by combinations ofof interferons fibroblasts and and ... Proost et al Introduction Chemokines form a family of chemotactic cytokines that are classified as CXC or CC according to the positioning of NH2terminal cysteines in their primary protein structure...

Ngày tải lên: 09/08/2014, 08:22

... progressively to the right as the induction time increased (Fig 5B) This shift, reflecting increases in the average amount of protein in responsive cells, is explained by protein accumulation over time ... 10 min, t ( protein ) = 20 min; Middle: t ( RNA ) = h, t ( protein ) 2 = h; Bottom: t ( RNA ) = h, t ( protein ) = 16 h 2 induction, most of the cells are still in the transcriptionally inactive ... the inducer shifts this single peak along the x-axis While observing protein expression in individual cells is informative for gauging the mode of gene induction, caution should be exercised in...

Ngày tải lên: 13/08/2014, 23:20

... without forming aggregates Many kinds of proteins are involved into the DNA compaction process Histone chaperons are a group of proteins in eukaryotic cells to help chromatin assembly The binding mechanisms ... explaining the DNA structure at large force ( >10 pN) In live cells, most DNA -protein interactions occur under the force of

Ngày tải lên: 12/10/2015, 17:34

... phosphorylated in response to convulxin (data not shown), suggesting that it may associate with GPVI in the absence of the FcR c-chain Convulxin also induced an increase in calcium in GPVI-expressing Jurkat ... chimeric protein (GPVI–GFP) into COS-7 cells Cellular localization of GPVI–GFP in COS-7 cells was analysed by immunohistochemistry Cells transfected with GFP or GPVI–GFP were incubated with convulxin ... c-chain was observed in cells expressing both wild-type GPVI and FcR c-chain (K562/F1-c), but not in cells expressing only the FcR c-chain (K562/pRc-c) (Fig 6A) This demonstrates that convulxin...

Ngày tải lên: 22/02/2014, 07:20

... encoding plasmids and 36 h postincubation the protein fraction was isolated from the cells and subjected to Western blotting analysis The extent of protein expression is much lower in the HEK293 cells ... protease Asp2, also called BACE protein or memapsin [9–13] This protein is involved in proteolytic cleavage of an amyloid precursor protein APP, and directly in uences the release of the toxic ... BACE protein, and in consequence, to reduce the amount of Ab peptide released in neuronal cells We asked the question whether we can inhibit expression of the gene of BACE protein using our engineered...

Ngày tải lên: 08/03/2014, 08:20

... activity of harmine in vivo Interestingly, our results show that harmine not only inhibits the protein serine ⁄ threonine kinase activity of the mature enzyme, but also the tyrosine autophosphorylation ... transfection, cells were incubated with indicated concentrations of harmine for 30 (E) HeLa cells were incubated in the presence of lM harmine where indicated for 30 min, followed by immunoprecipitation ... phosphorylation in U2OS cells Many protein kinases interact with their substrates in complexes that can be co-precipitated For example, caspase associates with CDK1–cyclin B1 in cells during G2 and...

Ngày tải lên: 30/03/2014, 02:20

... ferritin in HepG2 cells following IVI exposure for 0–24 h The decrease in the iron content of ferritin paralleled the increase in ferritin content itself (Fig 5) The faster the initial increase in ... removed by washing the cells with DMEM containing 50 lM DTPA and two more washings with DMEM alone IVI induced cell injury was assessed by measuring leakage of lactate dehydrogenase (LDH) into the ... impairment of synthesis of iron containing proteins or in cell injury by pro-oxidants [43], such findings in nonreticuloendothelial cells may have important implications in the generation of the adverse...

Ngày tải lên: 31/03/2014, 07:20

... Low) in HCV polyprotein expressing cells and in staurosporine treated cells, in contrast with cells infected with the VT7-HCV7.9 in the absence of IPTG or in uninfected cells These results indicate ... rounded in cells infected with VT7-HCV7.9 + IPTG, in comparison with uninfected cells or with cells infected in the absence of IPTG HCV polyprotein expression in human HeLa and HepG2 cells induces ... HCV proteins in VT7-HCV7.9 infected HeLa cells Immunogold electron microscopy analysis of the localization of HCV proteins in VT7-HCV7.9 infected HeLa cells HeLa cells infected with VT7-HCV7.9 in...

Ngày tải lên: 20/06/2014, 01:20

... fluorescence intensity was much stronger at a later time Figure shows that the fluorescence intensity increased almost linearly during the incubation period from 30 to 55 min, demonstrating a gradual increase ... CdTe QDs in living cells by means of confocal microscopy This report shows that QDs are localized not only in lysosomes, but also in Golgi complexes of the human hepatocellular carcinoma (QGY) ... QDs used in this work could be well internalized into living cells in vitro over a time period of about h This is probably due to the fact that the surface of the thiol-capped QD contains carboxylic...

Ngày tải lên: 22/06/2014, 00:20

... required staining Figure shows that even without staining this imaging method can readily explore in detail the dissimilarities in the internalization of different AuNPs by different cell lines Specifically, ... formed by aggregation of the internalized AuNPs, for 2D and 3D cultures of EMT and HeLa cell (without staining) The distributions were obtained by analyzing EMT cells and HeLa cells These results indicate ... Adsorption and Internalization by Cells Small 2010, 6:517-522 24 Chen J, Irudayaraj J: Quantitative investigation of compartmentalized dynamics of ErbB2 targeting gold nanorods in live cells by single...

Ngày tải lên: 11/08/2014, 00:23

... C-terminus domain, to expose binding sites for Src-homology-2 (SH2) domain-containing proteins and phosphotyrosine-binding (PTB) domain-containing proteins Adaptor proteins with a SH2-binding domain such ... epitope tag heparin-binding epidermal growth factor protein kinase C-related kinase homology region IQ motif containing GTPase activating protein infrared insulin receptor tyrosine kinase substrate ... techniques, including FCS and FCCS SW-FCCS had not been applied to study molecular interactions in live cells In this study we further applied SW-FCCS to study protein- protein interactions in vivo,...

Ngày tải lên: 14/09/2015, 14:25