RNF6 silencing attenuates DHT-induced K63-linked polyubiquitination of AR, and DHT upregulates its down-stream target sKit-L. (a) GCs from preantral and antral follicles were cultured with DHT (0 to 1 µm, 24 hours), subjected to immunoprecipitation with anti-AR, and followed by immunoblotting with anti-K48 ubiquitin or K63 ubiquitin. Results are expressed as means ± standard error of the mean (SEM) (n = 3) and analyzed by two-way ANOVA and Bonferroni post hoc test. **P < 0.01 versus CTL. (b) GCs were transfected with RNF6 siRNA (50 nM, 12 hours; scramble siRNA as control) and then treated with DHT (1 µm, 24 hours). Cells were immunoprecipitated using anti-AR, and the precipitate was immunoblotted with anti-K63 ubiqutin antibody. Results are expressed as means ± SEM (n = 3) and analyzed by two-way ANOVA and Bonferroni post hoc test. **P < 0.01, DHT versus DHT + RNF6 siRNA. (c) Changes in contents of sKit-L or mKit-L in GC cultures were assessed by Western blotting, expressed as means ± SEM (n = 3), and analyzed by two-way ANOVA and Bonferroni post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 versus DHT at 0 hours. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.