Western Blot Analysis of Cellular Proteins

As described previously, we extracted proteins from cells with RIPA lysis buffer, separated equal amounts of protein using SDS-PAGE, and then transferred the protein to a 0.45-µm PVDF membrane (Millipore, USA). Next, the membrane was blocked with 5% skim milk for 1 h at room temperature before it was washed 3 times with 1× TBST for 10 min each time. The membranes were then incubated with the following antibodies: rabbit anti-GAPDH (#5174), anti-Notch2 (#5732), anti-Bax (#0120), anti-Bcl-2 (#6139), anti-γH2AX (#8482), anti-Cyclin D1 (#0931), anti-AKT (#6261), anti-phospho-AKT (#0016), anti-mTOR (#6308), and anti-phospho-mTOR (#3308), all of which were purchased from Affinity Biosciences (OH, USA). A chemiluminescence reagent was used to visualize the protein bands, and the grayscale values of the bands were measured with Image Lab 6.0 software (Bio-Rad) [19 (link)].