Ecl solution

Protein samples were extracted using RIPA lysis (R0010, Solarbio) and quantified by a BCA Protein Assay Kit (PC0020, Solarbio). For the determination of protein level, an equal volume of protein samples was loaded on SDS‐PAGE gel, and then transferred onto PVDF membrane (IPVH00010, Millipore). After blocking in fresh skim‐milk, the membranes were incubated with primary antibodies overnight at 4°C and washed in TBST. Then corresponding secondary antibodies were utilized to conjugate the primary protein targets. Finally, the protein blots were visualized with ECL solution (PE0010, Solarbio), and the optical density was analyzed using Gel‐Pro‐Analyzer (Media Cybernetics).
All antibodies used in this study are listed as follows: PHLPP2 antibody (25244‐1‐AP, Proteintech), FOXO1 antibody (18592‐1‐AP, Proteintech), AKT antibody (#4691, CST), p‐AKT antibody (#4060, CST), Ki67 antibody (A11390, ABclonal), p21 antibody (10355‐1‐AP, Proteintech), p27 antibody (25614‐1‐AP, Proteintech), Cyclin D1 antibody (A0310, ABclonal), cleaved caspase‐3 (#9661, CST), cleaved caspase‐9 (#7237, CST), GAPDH antibody (60004‐1‐Ig, Proteintech), HRP‐conjugated goat anti‐rabbit antibody (SE134, Solarbio), and HRP‐conjugated goat anti‐mouse antibody (SE131, Solarbio). GAPDH was used as internal control.

Total protein of cells was extracted by using RIPA buffer containing protease inhibitors (Solarbio, Beijing, China). Protein concentration was quantified using Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China). The total proteins (30 μg for each) were separated using 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Following blocking with 5% skimmed milk at room temperature for 1 h, the membrane was incubated primary antibodies at 4 °C overnight. After incubation with primary antibodies, membranes were washed with TBST followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. These bands were detected using ECL solution (Solarbio, Beijing, China).