Figure 4: Ser42 phosphorylation affects Bmal1 transcriptional activity.
From: Insulin post-transcriptionally modulates Bmal1 protein to affect the hepatic circadian clock
(a) ChIP analysis of the occupancy of Ad-FLAG-WT-Bmal1 or Ad-FLAG-S42A-Bmal1 on the promoters of Dbp and Nr1d1 in primary hepatocytes co-infected with Ad-Clock (n=3, left). Immunoblotting analysis of relative FLAG-Bmal1 (FLAG) protein amounts in experiments with the corresponding conditions (right). (b) Transient luciferase reporter assay of Per1-Luc activity in HEK 293T cells co-transfected with Clock together with control vector, WT or indicated Bmal1 mutants (top, n=3). The amounts of nuclear FLAG-Bmal1 proteins in experiments with the corresponding conditions were shown by immunoblotting analysis at the bottom. (c) Quantitative PCR analysis of mRNA levels of Dbp and Nr1d1 in insulin-treated (50 nM, 1h) primary hepatocytes infected with Ad-FLAG-WT-Bmal1 or Ad-FLAG-S42A-Bmal1 adenoviruses (n=3). (d) Immunoblotting analysis of Dbp and Nr1d1 protein amounts in liver homogenates from mice tail-vein injected with Ad-FLAG-WT-Bmal1 or Ad-FLAG-S42A-Bmal1 and injected intraperitoneally with insulin (2 U kg−1) at ZT6 and then killed at ZT8, and corresponding densitometry analysis of relative Dbp, Nr1d1 and FLAG-Bmal1 protein amounts were shown on the right (n=3). Data are represented as mean ±s.e.m, statistical analyses were performed with a two-tailed unpaired Student’s t-test, *P<0.05, **P<0.01, #no significant difference.
