Fig. 4: CLOCK and BMAL1 block CUL3-mediated RHOA ubiquitin degradation.
From: CLOCK and BMAL1 stabilize and activate RHOA to promote F-actin formation in cancer cells
a, b Stability analysis of RHOA protein. a HeLa and HepG2 cells transfected with shNC, shBMAL1, or shCLOCK were treated with cycloheximide (20 μg/ml) for the indicated duration before harvesting. Expression of RHOA was detected by western blotting. b RHOA expression was quantified with ImageJ and then normalized to GAPDH expression. The protein level at 0 h was taken as 1. c Cells were treated as indicated, and the concentration of MG132 used was 20 μM. Data are presented as the mean ± sd (n = 3); *p < 0.05; **p < 0.01; n.s. not significant. d The ubiquitination levels of RHOA were analyzed by Co-IP assay. HeLa cells transfected with HA-ubiquitin (Ub) were treated with 20 μM MG132 for 6 h before harvesting. Data are presented as the mean ± sd (n = 3); *p < 0.05; **p < 0.01; n.s. not significant. e The efficiency of siCUL3 was tested with a western blot assay. siCUL3 #2 siRNA was selected for later experiments. f The complex was immunoprecipitated from total protein extracts collected from HeLa cells transfected with corresponding plasmids. The proteins were then analyzed by Co-IP with antibodies against CUL3. g The expression of RHOA was verified by western blotting in HeLa cells transfected with corresponding plasmids
