Ab183758

The cells were lysed in RIPA buffer supplemented with protease inhibitor and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Inc.). The protein concentration was then measured with a BCA Protein Assay Kit (Beyotime, Shanghai, China). The total proteins were separated with SDS-PAGE and then electrically transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk, the membranes were incubated with specific primary antibodies at 4C° overnight. The membranes were washed with TBST and incubated with a secondary antibody for 1.5 h at room temperature. Finally, the membranes were incubated in Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) before visualization. The primary antibodies used were as follows: mouse anti-GBAS (1:200, OriGene, TA505590, Rockville, MD, USA), rabbit anti-AKT1 (1:300, Abcam, ab183758, Cambridge, MA, USA), mouse anti-AKT2, rabbit anti-Bax (1:300, Abcam, ab175354 and ab32503, Cambridge, MA, USA), mouse anti-CHEK1 (1:300, CST, 2360, Danvers, MA, USA), and mouse anti-GAPDH (1:2000, Santa-Cruz, sc-32,233, Dallas, TX, USA). GAPDH was used as an internal reference.