Fig. 5: SEMA3G promotes c-Myc degradation in a WWP2-dependent manner.

A HEK-293 cells were transfected with Flag-tagged c-Myc and/or MYC-tagged WWP2 for 48 h. The interaction of c-Myc and WWP2 were detected using Co-immunoprecipitation. B The binding of endogenous WWP2 to c-Myc in GSC07 cells by co-immunoprecipitation analysis. C HEK-293 cells were transfected with Flag-tagged c-Myc domains and MYC-tagged WWP2 for 48 h. The interaction of c-Myc and WWP2 were detected using co-immunoprecipitation. D The overexpressed GSC07 cells (+) were transfected with WWP2-overexpresson lentivirus again (++) to induced the higher expression of WWP2. The protein level of c-Myc in the GSC07 cells with various WWP2 overexpression was determined by western blot. E Data summary of (D) are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by one-way ANOVA. F GSC07 cells were transfected with various WWP2 silence lentivirus for 72 h and screened using puromycin (1 μg/ml) to induced the stable knockdown cell. The protein level of c-Myc was determined by western blot. G Data summary of (F) are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by one-way ANOVA. H The WWP2 overexpressed GSC07 cells were treated with CHX (10 µg/ml) for the indicated time points. The protein level of c-Myc was determined using western blot. I Data summary of (H) are shown as mean ± s.e.m. ^**P < 0.01. Data were analyzed by two-way ANOVA. J The WWP2 overexpressed GSC07 cells were treated with MG132 (μmol/l) for 6 h. The protein level of c-Myc was determined using western blot. K Data summary of (J) are shown as mean ± s.e.m. ^**P < 0.01. Data were analyzed by one-way ANOVA. L The ubiquitination level of c-Myc in WWP2 overexpressed GSC07 was determined by co-immunoprecipitation analysis. M The WWP2 knockdown GSC07 and GSC27 cells were treated with or without SEMA3G for 72 h. The protein level of c-Myc was determined using western blot. N, O Data summary of (M) are shown as mean ± s.e.m. ^**P < 0.01, ^***P < 0.001. Data were analyzed by one-way ANOVA. Representative images (P) and the number (Q) of neurospheres of WWP2 knockdown GSC07 that were treated with or without SEMA3G (200 ng/ml) for 7 days. Images of neurospheres were captured using microscope. Scale bar, 100 μm. Data are shown as mean ± s.e.m. ^***P < 0.001. Data were analyzed by one-way ANOVA. All the western blot bands represent one of the three independent experiments.