Quantifying Cellular Protein Expression Using SDS-PAGE and Western Blotting

Total protein from cells was isolated using the radio-immunoprecipitation assay lysis buffer (P0013C, Beyotime Biotechnology Co., Ltd., Shanghai, China) containing phenylmethylsulfonyl fluoride. After concentration examination by a bicinchoninic acid kit (Thermo Fisher Scientific), an equal amount of protein sample (50 μg) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and loaded onto polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA, USA). After that, the membranes were blocked by 5% nonfat milk for 1 h and reacted with the antibodies against NRBP2 (1:1,000, PA5-65039, Thermo Fisher Scientific), GATA1 (1:1,000, ab181544, Abcam Inc., Cambridge, MA, USA) and GAPDH (1:2,500, ab181602, Abcam) at 4°C overnight, and then with goat anti-rabbit immunoglobulin G (IgG) (1:2,000, ab97051, Abcam) at 22–25°C for 1 h. The protein blots were visualized using an enhanced chemiluminescence kit (P0018FS; Beyotime Biotechnology Co., Ltd., Shanghai, China) and photographed by an image analyzing system (Bio-Rad Laboratories, CA, USA). The intensity of the protein signals was determined using the Quantum One v.4.6.2 software with GAPDH as the internal loading [20 (link)].