Cell pellets were washed twice with cold PBS (Gibco) and then lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 1% phenylmethanesulfonyl fluoride (PMSF; Beyotime Biotechnology) and 1% phosphatase inhibitor on ice. The protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology). Protein samples (10 μg) were separated by SDS-PAGE and transferred to PVDF membranes as previously described
[15] (link), and then incubated with anti-PSMA (1:1000 dilution; ab133579; Abcam, Cambridge, UK), anti-HIF1α (1:1000 dilution; ab51608; Abcam), and anti-GAPDH (1:2000 dilution; ab179467; Abcam) antibodies at 4°C overnight. Membranes were washed three times with TBST and incubated with the corresponding HRP-conjugated secondary antibodies (1:5000 dilution; 7076/7074; CST, Beverly, USA) at room temperature for 2 h. The membranes were washed again and then incubated with enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, USA) for 1 min. Finally, the protein band images were captured and analyzed.
[15] (link), and then incubated with anti-PSMA (1:1000 dilution; ab133579; Abcam, Cambridge, UK), anti-HIF1α (1:1000 dilution; ab51608; Abcam), and anti-GAPDH (1:2000 dilution; ab179467; Abcam) antibodies at 4°C overnight. Membranes were washed three times with TBST and incubated with the corresponding HRP-conjugated secondary antibodies (1:5000 dilution; 7076/7074; CST, Beverly, USA) at room temperature for 2 h. The membranes were washed again and then incubated with enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, USA) for 1 min. Finally, the protein band images were captured and analyzed.
