Kgp113k

Total cell extract were analyzed by Western blot as described previously (Jiao et al., 2018 (link)). Briefly, the total protein from treated cells was extracted using radio-immunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, Shanghai, China) supplemented with protease inhibitors (P1006, Beyotime) and phosphatase inhibitors (P1046, Beyotime). The protein concentrations were measured using a Bicinchoninic Acid (BCA) protein assay kit (P0010S, Beyotime). An equivalent of 20 micrograms of total protein extract were resolved by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (KGP113K, KeyGen Biotech. Co. Ltd., Nanjing, China), then the gels were transferred to PVDF membranes (Millipore, Temecula, CA, USA). Subsequently, the polyvinylidene difluoride (PVDF) membranes were blocked with 5% nonfat dried milk solubilized in TBST for 2 h and probed with primary antibodies against LC3, p62, p-S6 (Ser240/244), S6, p-P70S6K (Thr389), P70S6K, p-GSK3β, GSK3β, p-AKT (Ser473), AKT, cleaved caspase-3, Bax, Bcl-2, and β-actin overnight at 4°C. Then, the membranes were incubated with secondary antibodies for 2 h at room temperature. The immunoreactive bands were revealed using ECL Western blot protocol (P0018, Beyotime). The intensity of bands was measured using the Image Lab 5.0 software.

To detect the expression of specific proteins, 40 μg of total protein was separated on a SDS-polyacrylamide gel (KGP113K; KeyGEN, China) and wet transferred to a nitrocellulose transfer membrane (PALL66485F; VICMED, China). The membrane was probed with a primary and a secondary antibody. The protein bands were scanned and visualized using an Odyssey® Infrared Imaging System by LI-COR Biosciences (USA).