Figure 4.

The E3 ligase activity is required for the STUB1-mediated BMAL1 reduction. A, STUB1 expression down-regulates BMAL1 protein. HEK293T cells were transfected with the control or Myc-STUB1 plasmid, respectively. Cell lysates were immunoblotted for endogenous BMAL1, Myc-STUB1, and β-tubulin. The experiments were carried out three times, and means ± S.D. (error bars) were plotted with GraphPad Prism. p value was calculated using Student's t test. **, p < 0.01. B, gradient-increased transfection of STUB1 gradually reduces BMAL1 protein level. HEK293T cells were transfected with different amounts of the control or Myc-STUB1 plasmid in 6-well plates. Cell lysates were immunoblotted for endogenous BMAL1, Myc-STUB1, and β-tubulin. The experiments were carried out three times, and means ± S.D. were plotted with GraphPad Prism. Student's t test was used: *, p < 0.05; ns, not significant. C, the regulation of BMAL1 by STUB1 requires its E3 ligase activity. HEK293T cells were transfected with the control, WT STUB1, or H260Q STUB1 (inactive mutant) plasmid, and cell lysates were immunoblotted. Student's t test was performed for the data from triplicates, and means ± S.D. were plotted. ***, p < 0.001; ns, not significant. D, STUB1 knockdown increases BMAL1 protein level. HEK293T cells were transfected with control or STUB1 specific siRNAs, respectively, for 48 h using Lipofectamine 2000 transfection reagent. Cell lysates were immunoblotted with the indicated antibodies. The experiments were performed in triplicates, and means ± S.D. were plotted. Student's t test was used: ***, p < 0.001.