USP13 physically interacts with BMAL1 to regulate its ubiquitination. (A) M17 cell lysate was immunoprecipitated with anti-BMAL1 (Up panel), or anti-USP13 (Down panel), and analyzed by western blotting with anti-USP13. (B) Bacterial-purified GST or GST-USP13 was incubated with His-BMAL1 isolated from E. coli, and the GST pull-down assays were performed followed by western blotting analysis using anti-BMAL1. (C) M17 cells were transfected with pcDNA3.1 (Vector), pcDNA3.1/Flag-USP13 (USP13), or pcDNA3.1/Flag-USP13 C345A (USP13 CA) at indicated doses for 24 h, the cell lysates were analyzed by western blotting, and quantitated. (D) M17 cells were transfected with shUSP13-1 or shUSP13-2 for 48 h, and the cell lysates were analyzed by western blotting, and quantitated. (E) M17 cells were transfected with control (pll3.7) or shUSP13, then treated with cycloheximide (CHX) for the indicated time, and the cell lysates were analyzed by western blotting with quantitation and compared by two-way RM ANOVA. (F) HEK-293T cells were transfected with indicated ectopic vectors for 48 h, and the cell lysates were immunoprecipitated with Ni-NTA beads, then analyzed by western blotting with BMAL1 antibody. The whole cell lysate of each treatment was also used to determine the expression of indicated proteins. (G and H) M17 cells were transfected with indicated ectopic vectors and analyzed by western blotting, and quantitated. (I) 8-wk-old C57BL/6 mice were icv-injected with indicated AAV virus bilaterally. 18 days later, the hypothalami of mice were isolated before being subjected to detect the ubiquitin-conjugated BMAL1 and indicated protein expression in the tissue, with the expression of indicated proteins quantitated in each group (n = 3). The unpaired t test was used in comparisons in C, D, and G–I. *P < 0.05; **P < 0.01; ***P < 0.001. n.s, no significance. Source data are available for this figure: SourceData F6.