Fig. 5

USP7 regulates ARF4 stability through K48-linked deubiquitination

A, B. After transfection of SHG-140 cells with shUSP7 for 24 h or treatment of cells with P5091 (2 µM) for 48 h, cells were treated with CHX (100 µg/ml) at different times and ARF4 expression was analyzed by western blotting, n = 3. C, D. SHG-140 cells were transfected with shUSP7 for 24 h or treated with P5091 (2 µM) for 48 h followed by treatment with the proteasome inhibitor MG132 (10 µM) for 6 h. ARF4 expression was analyzed by western blotting, n = 3. E, F. SHG-140 cells were transfected with shUSP7 for 24 h or treated with P5091 (2 µM) for 48 h followed by treatment with MG132 (10 µM) for 6 h. The protein extracts were immunoprecipitated with IgG beads of anti-ARF4, and then ubiquitin and ARF4 expression were detected by western blotting. G, H. SHG-140 cells were transfected with shUSP7 for 24 h or treated with P5091 (2 µM) for 48 h followed by treatment with MG132 (10 µM) for 6 h. The protein extracts were immunoprecipitated with IgG beads of anti-ARF4, and then the expression of K48-ubiquitin, K63-ubiquitin and ARF4 was detected by western blotting. I A proposed mechanism for USP7 to regulate the ARF4 level in GBM. All statistics are expressed as mean ± S.E.M., ^#P = NS, *P < 0.05,**P < 0.01 or ***P < 0.001