Anti phospho akt

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)].

Western blot analysis was performed as described in our previous study [13 (link)]. Briefly, equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, USA). After the membranes were blocked by TBST (TBS plus 0.05% Tween 20) plus 1% nonfat dry milk for 60 min, proteins were incubated with primary antibody overnight at 4°C. The following antibodies were used: anti-IR (1 : 1000; Affinity biosciences, USA), anti-phospho-IR (1 : 2000; Affinity biosciences, USA), anti-IRS-1 (1 : 1000; Cell signaling, USA), anti-phospho-tyr-IRS-1 (1 : 1000; Cell signaling, USA), anti-PI3K (1 : 1000; Affinity biosciences, USA), anti-phospho-PI3K (1 : 2000; Affinity biosciences, USA), anti-AKT (1 : 1000; Cell signaling, USA), anti-phospho-AKT (1 : 2000; Affinity biosciences, USA), anti-MCT4 (1 : 2000; Proteintech, USA), anti-GLUT4 (1 : 1000; Proteintech, USA), and anti-GAPDH (1 : 1000, Cell signaling, USA). PVDF membranes were then washed by TBST and incubated with HRP-linked antibody(1 : 3000; Cell signaling, USA) at room temperature for 60 min. PVDF membranes were then washed by TBST and developed using Enhanced Chemiluminescence Reagents (ECL; New Cell & Molecular Biotech, China). At last, the protein bands were analyzed by the Image Lab system.