Anti gapdh antibody

Total proteins were extracted from fresh tissues and cell lines with RIPA lysis buffer containing a cocktail of protease inhibitors (Beyotime Institute of Biotechnology). The protein samples were then separated by 10% SDS-PAGE and electrotransferred to nitrocellulose membranes (Millipore). After blocking with 5% nonfat milk dilution with TBST for 1 h at room temperature, the membrane was incubated with primary antibodies against INPP4B, p-SGK3^T320, SGK3, p-AKT^T308, and AKT at 4°C overnight. After washing with TBST, the membranes were incubated with the secondary antibody (Cell Signaling Technology) at a dilution of 1:3000 at room temperature for 60 min. The membranes were then washed with TBST, and the bound antibodies were detected with an enhanced chemiluminescence system. Anti-GAPDH antibody (1:3000; Vazyme) was used as a loading control.

Total proteins were extracted from fresh frozen tissues and cell lines by RIPA lysis buffer. The protein concentration of the supernatant was detected by the BSA Protein Assay Kit (Beyotime institute of Biotechnology, Jiangsu, China). The protein samples were separated on 8% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes (Millipore, Billerica, MA). After blocking with 5% nonfat milk dilution with TBST (tris-buffered saline with tween-20) for 1 h at room temperature, the membranes were incubated with rabbit anti-RBMS3 antibody (1:1000; Abcam) and rabbit anti-HIF1A antibody (1:1500; Abcam) at 4°C overnight. After washing 3 times with TBST per 10 min, the membranes were then incubated with horseradish peroxidaselabeled anti-rabbit IgG as the secondary antibody (Epitomics) at room temperature for 60 min. Afterwards, after 3 times washing with TBST per 10 min, the membranes were detected with the enhanced chemiluminescence system. Anti-GAPDH antibody (1:3000; vazyme) was used as a loading control.