Anti p85α

Cells were lysed and protein extracted with RIPA buffer (Thermo Fisher Scientific). Then, 30 μg of total proteins was separated by 10%‐15% SDS‐polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (EMD Millipore). After blocking with 5% fat‐free milk solution, the protein of interest was detected by using a primary antibody and a corresponding HRP‐linked secondary antibody. The primary antibodies used were as follows: anti‐Runx2 (0.4 μg/mL), anti‐eNOS (0.5 μg/mL) from Santa Cruz Biotechnology; anti‐β‐actin (0.4 μg/mL) from Sigma‐Aldrich; anti‐osteopontin (OPN) (1 μg/mL) from Bioworld Technology; anti‐pAktS473 (1:2000), anti‐pAktT308 (1:1000), anti‐Akt (1:1000), anti‐Rictor (1:1000), anti‐RheB (1:1000), anti‐p85α (1:1000), anti‐Calponin‐1(1:1000), anti‐PARP‐1 (1:1000), anti‐Histone H3(1:2000), anti‐Bcl‐XL(1:1000), anti‐Bim(1:1000), anti‐Bax(1:1000), anti‐pS6K(1:1000), anti‐S6K(1:1000) from Cell Signaling Technology; anti‐pFOXO3a (1:2000), anti‐FOXO3a (1:1000), anti‐α‐SMA (0.5 μg/mL) from Abcam, anti‐caspase 3 (1:2000) from Cell Signaling Technology. and anti‐GAPDH (1 μg/mL) from GeneTex. The blots were developed with an enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific) and exposed to X‐ray film to obtain optimal results in the dark room. The density of bands was quantified by Image‐Pro Plus 6.0 software.