Protein phosphatase inhibitor

Cell lysis was performed as previously described with minor modifications (46 (link), 48 ). In brief, cells were lysed on ice in cytoplasmic lysis buffer (50 mM Tris [pH = 8.0], 150 mM NaCl, and 0.5% Triton X-100) with 1% protein/phosphatase inhibitor (Thermo Fisher Scientific; catalog no.: 78442). The cell suspension was centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was transferred to a new tube and labeled the cytoplasmic fraction. Remaining cell pellet was washed 2× in cytoplasmic lysis buffer, and supernatant was discarded. Pellet was then resuspended in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH = 8.0], 150 mM NaCl, 0.1% SDS, 1% Triton X-100, and 0.5% sodium deoxycholate) with 1% protein/phosphatase inhibitor (Thermo Fisher Scientific; catalog no.: 78442), and the suspension was sonicated and then clarified by centrifugation at top speed. Supernatant was transferred to a new tube and labeled the nuclear fraction. Subcellular fractionation was confirmed via Western blot showing isolation of GAPDH and H3 to cytoplasm and nucleus, respectively.

The expression of Bcl-2, Bax, Caspase-3, PI3K, Akt, phospho-PI3K, and phospho-Akt was assessed by western blot analysis. Cell samples were lysed on ice with lysis buffer containing cocktail proteinase inhibitors and protein phosphatase inhibitors (Thermo Fisher Scientific, Inc., Rockford, USA). The protein concentration was quantified using a bicinchoninic acid (BCA) assay kit (Beyotime, Shanghai, China). Protein samples were separated by SDS-PAGE using 4-20% precast gradient polyacrylamide gels (Shanghai Suolaibao Bio-Technology Co., Ltd., Shanghai, China). After separation by SDS-PAGE, proteins were transferred to a PVDF membrane. The blots were then incubated with primary antibodies and subsequently incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies. The results were detected using G:Box Chemi XRQ imaging system (Cambridge, Britain).