Western Blot Analysis of SRD5A2 Mutants

Western blot was performed as previously described [33 (link)]. Briefly, after HEK293 cells has been transfected with wild-type or p.H232R mutant SRD5A2 plasmids for 48 h, they were collected from six-well plates and lysed with RIPA buffer (P0013; Beyotime Biotechnology). The whole-protein lysates were separated by 12% SDS-PAGE and then transferred to nitrocellulose membranes. After blocking with 5% milk, the membranes were incubated with the primary antibodies monoclonal anti-flag (F9291; Sigma) and β-actin (sc-69879; Santa) at 4°C overnight. After rinsing with Tris-buffered saline containing 1% Tween-20 (TBST), the membrane was then incubated with appropriate HRP-conjugated secondary antibodies (sc516102; Santa Cruz) and detected with an ECL Plus kit (P1050; Applygen).