Gapdh mouse

ccRCC tumor tissues or cultured cell were homogenized in lysis buffer containing protease inhibitors, centrifuged at 12,000 g for 15 min at 4°C, and the protein concentration in the supernatant was determined with a bicinchoninic acid assay kit (Beyotime Institute). The precipitated proteins (80 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for 2 h at 75V and transferred to polyvinylidene difluoride (PVDF) membranes on wet transfer equipment at 350 mA for 2 h. The membranes were blocked with 5% skim milk in TBS for 1 h at room temperature and then incubated overnight with rabbit polyclonal anti-TNIP1 (LSBio, USA, 1:1000), rabbit polyclonal anti-C/EBPβ (GeneTex, 1:1000), rabbit polyclonal anti-B-cell lymphoma 2 (Bcl-2, Proteintech, 1:500), rabbit monoclonal anti-Bcl-2-associated X (Bax, Proteintech, Chicago, USA, 1:1000), or rabbit monoclonal anti-cleaved-caspase 3 (Cell Signaling Technology, Inc., Danvers, MA, USA, 1:1000) primary antibodies at 4°C. Proteins were visualized by incubation with horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit IgG antibody (ZSGB-BIO, China, 1:5000) for 1h. Color was developed by electrochemiluminescence (ECL) and the images were scanned with an Alpha Innotech gel imaging system (Bio-Rad) and captured. GAPDH (mouse, 1:1000, Santa Cruz Biotechnology) was used as an internal control.