Journal: Journal of biological rhythms
Article Title: The E3 ligases Spsb1 and Spsb4 regulate RevErbα degradation and circadian period.
doi: 10.1177/0748730419878036
Figure Lengend Snippet: Representative western blot illustrating the stability of A) Flag-RevErbα or B) Flag-RevErbβ when co-expressed with Sport6 or Spsb1–4 in a CHX-chase experiment (hrs = time with CHX). C) Quantitation of western blot data from experiments of Flag-RevErbα or Flag-RevErbβ normalized to GAPDH and plotted relative to the time 0 point. Data are mean +/− SEM of n = 3–7 independent experiments for Flag-RevErbα and n=3 independent trials for Flag-RevErbβ except for Spsb3 (n=2)). Two-way ANOVA reveals a significant time x E3 ligase interaction on RevErbα stability for Spsb1 and Spsb4 (p<0.001) but not Spsb2 or Spsb3 (p>0.5). * = differences at individual time points between Spsb1/Spsb4 and the Sport6 control (p<0.05) using Sidak’s multiple comparison test. There was no significant effect of Spsb E3 ligases on Flag-RevErbβ stability (p>0.05 for all Two-way ANOVA outputs).
Article Snippet: Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen),1x non-essential amino acid (Invitrogen), and Penicillin/Streptomycin/ glutamine mix and cultured at 37°C in 5% CO 2 ( DeBruyne et al., 2015 ).HA-tagged Spsb2 (Cat# HG14695-CY) and Spsb3 (Cat# HG16867-CY) plasmids were obtained from Sino Biological.
Techniques: Western Blot, Quantitation Assay