The cells were harvested in radioimmunoprecipitation assay (RIPA) lysis buffer (Solaibio, cat#: R0020, China) supplemented with protease and phosphatase inhibitors (Protease Inhibitor PMSF, Solaibio, cat#: R0020, China and PhosSTOP, Roche, cat#: 04906845001, Switzerland). The protein concentration was measured using a BCA Protein Assay kit (Solarbio, cat#: PC0020, China). The protein samples were mixed with 4× sample buffer (Solarbio, cat#: P1015, China) and boiled for 5 min at 95°C for denaturation. Western blotting was performed according to standard protocols. The samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 60 V at RT) and transferred onto polyvinylidene difluoride (PVDF) membranes (90 V at 4°, 90 min). Then, the membranes were blocked with 5% non-fat dry milk, dissolved in TBST buffer for 2 h, and incubated with the primary antibodies (AR, 1:2,000, rabbit monoclonal, cat#:ab133273, Abcam; AKT, 1:1,000, rabbit monoclonal, cat#:4691, Cell Signaling Technology; p-AKT, 1:2,000, rabbit monoclonal, cat#: 4060,Cell Signaling Technology; GAPDH, 1:1,000, rabbit polyclonal, cat#: AB_2619673, ABclone Technology) overnight at 4°C. Next, the membranes were rinsed with TBST buffer (3 times, 5 min each) and incubated with the secondary antibody ((rabbit IgG (H&L) Antibody Dylight™ 800 Conjugated, cat#: 610-445-002, dilution 1:10,000) at RT in the dark for 2 h. The membranes were rinsed again, and the results were analyzed by an Odyssey IR fluorescence scanning imaging system (LI-COR, USA). Both the incubation and washing processes were carried out on a shaker table.

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