A Circadian Clock Translational Control Mechanism Targets Specific mRNAs to Cytoplasmic Messenger Ribonucleoprotein Granules
Description
This collection contains original, scanned and annotated western blots of the representative images shown in the supplementary files of the Cell Reports article, "A Circadian Clock Translational Control Mechanism Targets Specific mRNAs to Cytoplasmic Messenger Ribonucleoprotein Granules by Castillo et al."
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Protein was extracted as previously described (Jones et al., 2007) with the following modification. The extraction buffer contained 100 mM Tris-HCl pH 7.0, 1% SDS, 10 mM NaF, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM β-glycerophosphate, 1X aprotinin, 1X leupeptin hemisulfate salt, and 1X pepstatin A. Protein concentration was determined using a NanoDropTM Microvolume Spectrophotometer. Protein samples (100 µg) were separated on 10% SDS-PAGE gels and blotted to an Immobilon-P nitrocellulose membrane according to standard methods. The levels of P-eIF2α were detected using rabbit monoclonal anti-EIF2S1 (phospho S51) antibody) diluted 1:5000 in 5% bovine serum albumin, 1X TBS, 0.1% Tween, and anti-rabbit IgG HRP secondary antibody diluted 1:10000. Total eIF2α levels were detected using rabbit polyclonal anti-EIF2S1 antibody diluted 1:5000, and anti-rabbit IgG HRP secondary antibody diluted 1:10000. FRQ protein was detected using mouse monoclonal anti-FRQ antibody (gift from Dr. Michael Brunner) diluted 1:200 in 7.5% milk, 1X TBS, 0.1% Tween and anti-mouse IgG-HRP secondary antibody diluted at 1:10000 (Gorl et al., 2001). VVD::V5 was detected using mouse monoclonal anti-V5 antibody diluted 1:5000 in 5% milk, 1XTBST, 0.1% Tween, and anti-mouse IgG HRP secondary antibody diluted 1:10000. All proteins except FRQ were detected using SuperSignalTM West Pico PLUS Chemiluminiscent Substrate. FRQ was detected using SuperSignalTM West Femto Maximum Sensitivity Substrate. Densitometry was performed using NIH ImageJ software (Schneider et al., 2012) and normalized to protein loading using amido black-stained protein.