Western Blot Analysis of Protein Expression

The cells were inoculated in 100 mm culture dishes and incubated for 48 h after drug treatment. After sufficient lysis of the cells, the supernatant was collected by centrifugation. The protein content was determined using the BCA kit (Sigma-Aldrich). SDS-PAGE electrophoresis was performed to separate proteins with different relative molecular masses, which were then transferred onto PVDF membranes (Thermo Fisher Scientific). The proteins were blocked with 5% skim milk for 2 h, washed 3 times with TBST, and incubated with primary antibodies (GPX4, AR, FKBP51, PSA, LK2, S100P, TMPRSS2, Bax, caspase-3, Bcl-2, GPX1, AR, PSA, 1:1000, all purchased from Abcam) overnight at 4°C. PVDF membranes were washed with TBST, and peroxidase-labeled secondary antibody (1:10000, Abcam) was added, incubated for 2 h at room temperature, and washed 4 times with PBST. The proteins were visualized by a DAB immunohistochemistry color development kit (Sangon Biotech, China) and photographed using a gel imaging analysis system (Thermo Fisher Scientifi). Relative protein levels were calculated by ImageJ as previous report (Sun et al., 2022 (link)).