electroporation based methods for analysis of zebrafish brain development

... commissure Chapter – Electroporation- based methods for analysis of zebrafish brain development 3.1 Overview In order to investigate mechanisms of habenular commissure development, we required ... Formation of the habenular commissure 21 ii 2.9 CONCLUSIONS 22 2.10 EXPERIMENTAL PROCEDURES 22 CHAPTER – ELECTROPORATION- BASED METHODS FOR ANALYSIS OF ZEBRAFISH ... population of neurons This chapter presents a suite of techniques developed or optimized for the study of neural development in zebrafish Electroporation is a technique for the introduction of nucleic...

Ngày tải lên: 14/09/2015, 10:34

... 1.7.1.3: Immunofluorescence analysis for TJ protein localization 68 1.7.2: Analysis of TJ function 69 1.7.2.1: Analysis of fence function 69 1.7.2.2: Analysis of gate function ... isoforms Fig 13 shows two of the major protein complexes that occur in TJs Of the JAM-interacting proteins, Par-3 strongly suggests a role for JAM in TJ formation since Par-3 is a component of ... claudins, exogenous expression of JAM-A in L-cells does not induce TJ strand formation (Itoh et al., 2001) Evidence for a role of JAM proteins in the formation of TJ and cell polarity came from...

Ngày tải lên: 16/09/2015, 08:31

... has been established for Arabidopsis and rice [2–4] However, although plants encode for a large number of BTB proteins, a functional role has only been assigned for a few of them, including ETO1 ... interactors for the MATH domain, but would also provide information on to what extent the two different MATH domains of BPM1 and BPM3 target the same group of proteins (see Table S1A,B for identity ... expression strength ⁄ intensity: For BPM1 and BPM3 twofold, and for BPM4 threefold, the amount of RT-PCR product was loaded (compared with actin2 control reaction) (B) RT-PCR analysis showing BPM1, BPM2,...

Ngày tải lên: 18/02/2014, 06:20

... Hsp27 Hdj-2 is an isoform of Hdj-1 that has been previously shown not to decrease htt inclusion body formation in COS-7 cells [39] Immunoblot analysis of the intracellular level of Hsps revealed ... the percentage of cells presenting inclusions as well as the distribution of the size of the inclusions were dependent on when the analysis was performed after transfection Therefore, all the ... analyzed the activity of the Hsp70 ⁄ Hdj-1 reconformation machine by cotransfecting COS-7 cells with vectors encoding Hsp70 and the nonactive isoform of Hsp40, Hdj-2 In the presence of Hsp70 ⁄ Hdj-2,...

Ngày tải lên: 19/02/2014, 06:20

... identifying new classes of antibiotics displaying diverse mode(s) of action: antimicrobial peptides are currently considered to be some of the most promising candidates for the development of novel anti-infective ... (µM) % of lactamase release B 100 80 60 40 20 10 32 15 20 Time (min) Fig Permeation of E coli OM by Esc(1–18) (A) Effects of different concentrations of Esc(1–18) on permeation of the OM of E coli ... Permeation of the bacterial IM To assess the ability of Esc(1–18) to alter the permeability of the IM of E coli, · 106 cells were mixed with lm SYTOX Green in NaCl ⁄ Pi for in the dark After addition of...

Ngày tải lên: 16/03/2014, 00:20

... 150 V for  h For autoradiography, wet gels were exposed for h to a Kodak X-Omat film at )80 °C To ensure the complexes were formed by the association of proteins and nucleic acids, lg of each ... not part of any of the three LaGT complexes The values estimated for the protein masses included the mass of the Tel6 oligonucleotide ( 5.6 kDa) The binding specificity of LaGT protein-forming ... bands of  35 kDa and  52 kDa and the UV-irradiated complex of  18–20 kDa were in-gel digested with trypsin and subjected to MALDITOF MS and ESI-MS/MS analysis The MALDI-TOF spectra obtained for...

Ngày tải lên: 23/03/2014, 13:20

... 25S formation in Dnop53 ⁄ A-NOP53, compared with the much less affected formation of mature 18S rRNA (Fig 4B) Analysis of pre-rRNA and rRNA steady-state levels by means of northern blot was performed ... rRNA Analysis of rRNA processing showed that depletion of Nop53p leads to an accumulation of the 27S and 7S pre-rRNAs, confirming a role for Nop53p on late steps of processing Accumulation of unprocessed ... was performed after incubation of pmol of in vitro transcribed, uniformly labeled 5.8S rRNA with increasing amounts of His–Nop53p or bovine serum albumin (NEB), in the absence or presence of cold...

Ngày tải lên: 20/02/2014, 01:20

... 14-3-3 binding [36] We therefore suggest that analysis of the regulation by phosphorylation/14-3-3 proteins of NR or GS in crude fractions be performed in the presence of >1 mM NaF, or after passing ... MacKintosh et al., 1995) Assays were performed in the presence of 12 mM EDTA (d), mM ATP (j) or the absence of ATP (h) Similar profiles were seen for extracts of spinach leaves and cultured Arabidopsis ... found to catalyse the production of AMP from NADH, ATP and FAD (Table 3) Ninety percent of the amount of NADH used in our standard NR assay, and 60% of the amount of ATP in a GS assay, was converted...

Ngày tải lên: 08/03/2014, 08:20

... Ssa1p binds a hexameric form of Ure2p in the presence of ATP, whereas the form that is bound in the presence of ADP is different, and probably dimeric [6] We therefore performed Ure2p and Ssa1p ... assembly of Ure2p into fibrils [33] Thus, the binding of Ssa1p in the vicinity of this stretch interferes with Ure2p assembly into fibrils either because of a change in the conformation of this ... with 5% formic acid at 37 °C for 15 The extracted peptides were vacuum dried, dissolved in 1% formic acid and stored at )20 °C until MS analysis High mass MALDI-TOF MS High-mass MALDI-TOF mass...

Ngày tải lên: 15/03/2014, 00:20

... at °C for h For analysis of endogenous Brox, the total cell lysates of HEK293 cells cultured in DMEM containing 5% fetal bovine serum supplemented with FTI-277 to a final concentration of 10 lm ... overexpression of Strep-tagged Brox proteins Treatment of cells with FTI-277 caused a shift in the mobility of Strep–BroxWT to that of Strep–BroxC408S, but the treatment did not affect that of Strep–BroxC408S ... Overexpression of an ATPase-defective mutant of AAA type ATPase SKD1 (also named Vps4B), SKD1E235Q, is known to induce formation of abnormal endosomes As shown in Fig 7, a subset of punctate fluorescence...

Ngày tải lên: 16/03/2014, 06:20

... of prion diseases has remained uncertain In the most accepted ‘proteinonly’ hypothesis, the causative agent, the pathologic prion protein (PrPSc), is proposed to be a conformational isoform of ... sensitive assay for the detection of IgG binding to native PrPC, fluorescence-activated cell sorting (FACS) analysis of PC-12 rat pheochromocytoma cells, which express low levels of PrPC on their ... wild-type BPV-1 L1–VLPs were further analyzed for the presence of PrP or BPV-1 L1 epitopes For western blot analysis, PrP–VLPs, wild-type BPV-1 L1-VLPs or lysates of Sf9 cells infected with recombinant...

Ngày tải lên: 16/03/2014, 11:20

... acids of the Rabaptin-5c isoform, bearing a natural deletion of amino acids 22–64 of Rabaptin-5 [23], was able to interact with Rab4, thus demonstrating the dispensability of these amino acids for ... splicing from a single pre-mRNA [23] One of the recently identified Rabaptin-5 isoforms is Rabaptin-5d [23] Compared to Rabaptin-5, this isoform has a short deletion of 40 amino acid in the N-terminus ... Rabaptin-5 and its isoform (E-Rn5’s) (A,B), and FLAGÒ-tagged Rabaptin-5 and its isoform (Rn5-FLAGâ) (C) GTP in vitro, Rabaptin-5 alone could not support biological activity of Rab5 in early endosome...

Ngày tải lên: 16/03/2014, 18:20

... addition of small aliquots (1–5 lL) of a stock solution of the peptide to 0.5 mL of solution containing F-actin at a concentration of 2.5–4.0 mgÆmL)1 The broad signals of the spectrum of F-actin ... significance for understanding the role of TnI in the regulation of contraction in striated muscle Some aspects of this work have been briefly described in abstract form [26] MATERIALS AND METHODS ... the presence of the latter protein Evidence is provided for the induction of conformational changes in at least two regions of the actin molecule on binding the inhibitory domain of TnI to a third...

Ngày tải lên: 17/03/2014, 10:20

... was performed at 65 °C for and 37 °C for 60 followed by denaturation at 95 °C in a total volume of 50 lL of reaction mixture using the RT-PCR kit (Stratagene) PCR amplification was performed using ... endogenous WD gene Amplification conditions were: 95 °C for followed by 18 cycles of 95 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s, and 72 °C for Ó FEBS 2002 2154 W J Oh et al (Eur J Biochem 269) ... intensities of the amplified fragments were quantified by densitometric analysis As an internal control 791 base pairs of a-actin was amplified using forward and reverse primers The primers used for competitive...

Ngày tải lên: 17/03/2014, 23:20

... High Performance column (1.6 · 11 cm) This column was developed with a linear gradient of NaCl in the imidazole buffer (0–0.4 M, mLÆmin)1) For the purification of triflin, 500 mg of the venom of Trimeresurus ... procedures), we obtained mg of triflin and mg of latisemin from 500 mg of crude venom, respectively The predicted amino-acid sequences of triflin and latisemin are homologous to that of ablomin (83.7 and ... proposed for these family proteins Although the identity of the CAP proteins vs ablomin are relatively low, i.e 42.7% for acidic epididymal glycoprotein, 26.9% for antigen 5, 27.0% for glioma...

Ngày tải lên: 18/03/2014, 01:20

... addition of 125 lM spermine (Fig 2B) NMR was used for analysis of the effects of polyamine– phosphate interaction on the molecular arrangement of polyamines In Fig 3, the NMR-spectra of the single ... considered significant at P < 0.05 RESULTS GPC of nuclear extracts of replicating cells A representative profile of GPC analysis of the nuclear extracts of Caco-2 cells stimulated to replicate by ... GPC analysis performed on the nuclear extracts of Caco-2 cells starved for days revealed a very low < 1000 Da peak at the initial conditions (0 h), and the retarded and scarce formation of 4800...

Ngày tải lên: 23/03/2014, 21:21

... 0.25 µg of linearized DNA, µl of 5X T7 transcription buffer, 1.0 µl of 0.1 M DTT, 0.5 µl of SUPERase·In™ ribonuclease inhibitor (20 U/ µl) (Ambion, Austin, TX), 2.5 µl of an NTP Page of 11 (page ... Jonard G: High resolution analysis of the readthrough domain of beet necrotic yellow vein virus readthrough protein: a KTER motif is important for efficient transmission of the virus by Polymyxa ... studying gene silencing J Virol Methods 2003, 111(1):37-42 Scholthof HB: Rapid delivery of foreign genes into plants by direct rub-inoculation with intact plasmid DNA of a tomato bushy stunt virus...

Ngày tải lên: 18/06/2014, 22:20

... ml of Tris – HCl buffer, g of CsCl and 0.25 ml of phage suspension were then added to the vial This was centrifuged in a SW-50 rotor at 35,000 rpm for 48 hours to produce fractions An aliquot of ... microscopy of viruses Bioch Biophys Acta 1959, 34:103-110 Adams MH: Methods of Study of Bacterial Viruses In Bacteriophages Interscience Publishers, Inc New York; 1959:443-457 Adler J: A method for ... vulgaris, as in the case of other bacteria, have been utilized for typing schemes [20-22] and are structurally similar to phage from other The adsorption of phage PV22 on the surface of C jejuni flagella...

Ngày tải lên: 20/06/2014, 01:20

... gradient Inhibition of virion production by overexpression of fragments of endophilin If endophilins are important for virion production, the overexpression of fragments or of wild-type endophilin ... incorporation of any one of the various proteins involved in vesicular trafficking may depend on the behavior of the particular complex in which it resides; one explanation for the lack of incorporation of ... SP: Genetic assay for multimerization of retroviral gag polyproteins J Virol 1992, 66:5157-5160 Li X, Yuan B, Goff SP: Genetic analysis of interactions between Gag proteins of Rous sarcoma virus...

Ngày tải lên: 06/08/2014, 18:20