Ecl plus kit

Cells were collected after treatment, and the total protein was extracted in a cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). After the protein concentration was determined using the BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China), equal amounts of protein samples were electrophoresed on a sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) (12% (w/v) polyacrylamide gel) and transferred to a polyvinylidene fluoride (PVDF) membranes (Pall Corp., Port Washington, NY, USA). Thereafter, the membranes was blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. This process was followed by an overnight incubation with corresponding primary antibodies in TBST at 4 °C. The membranes were then washed thrice in TBST buffer, followed by incubation with 1:2000 dilutions of HRP-conjugated secondary antibody for 1.5 h at room temperature. The membranes were washed again three times in TBST. The proteins were visualized using the enhanced chemiluminescence (ECL) plus kit (Applygen Technologies Inc., Beijing, China). The membranes were then scanned using a Bio-Rad ChemiDoc XRS imaging system; the density of the bands on the blots was quantified by Gel-Pro Analyzer 4.0 software (Media Cybernetics, Rockville, MD, USA).

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).