P ampk thr172

The whole proteins in each cell samples were extracted by RIPA Lysis Buffer containing 1 mM PMSF (Beyotime). The protein was blocked and incubated with the primary antibodies against beclin-1, p62/SQSTM1 and LC3 (MBL; 1:1000), caspase 3, GAPDH, P-Akt (Ser473), P-AMPK (Thr172), P-ULK1 (Ser317) and P-mTOR (Ser2448) (Cell Signaling; 1:1000), overnight at 4 °C, respectively. The protein was then incubated with HRP-conjugated secondary antibody (1:5000) for 1 h at room temperature. The protein was detected using the eECL Western Blot Kit (Beyotime).

Protocols for immunoblotting and co-immunoprecipitation (IP) were as previously described^{5 (link)}. Antibodies were from the following sources: Cyclin B1, CDK1, phospho-histone H3 Ser10, pAMPK-Thr172, AMPK, Mcl-1, Bcl-2, Bcl-XL, pBcl-XL-Ser62 and cleaved PARP were from Cell Signaling Technologies. BAD, tubulin and BAX conformational antibody clone 6A7 were from Sigma-Aldrich. BAX was from Santa Cruz Biotechnology, vimentin was from Abcam, and cleaved caspase-3 was from Enzo Life Sciences.

Cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.5% Na-deoxycholate, 1% NP-40) supplemented with 1% phosphatase and 1% protease inhibitor cocktails, 5 mM NaF and 1 mM PMSF. Gel electrophoresis was performed according to standard protocols
[24 (link)]. Antibodies and working dilutions for western blot: AR (1:100, Genetex, Irvine, CA, USA), GAPDH (1:100,000, Millipore, Billerica, MA, USA), AMPK and p-AMPK-Thr¹⁷² (1:1000, Cell Signalling, Danvers, MA, USA), MID1 (1:400, Sigma-Aldrich), α4 (1:500, Abcam, Cambridge, UK), N-flag (1:1000, Sigma-Aldrich), PP2A (1:1000, Millipore). Immunoblot bands were scanned and quantified using a scanning densitometer (Odyssey; Li-Cor Biosciences, Lincoln, NE, USA). The housekeeping protein GAPDH served as loading control.

Cells were lysed with RIPA lysis buffer (Solarbio Biotechnology Beijing, Beijing, China) supplemented with 1 mM protease and phosphatase inhibitors. Protein concentrations were measured using the BCA Protein Assay Kit (Solarbio Biotechnology Beijing, Beijing, China), separated with 6–15% SDS polyacrylamide gels, and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1–2 h at room temperature and incubated at 4 °C overnight with different primary antibodies: AMPK (1:1000, Wanleibio, Shenyang, China, WL02254), P-AMPK (Thr 172) (1:2000, Cell Signaling Technology, Danvers, MA, USA, #2531), P-ACC (Ser79) (1:2000, Cell Signaling Technology, Danvers, MA, USA, #3661), ACC (1:2000, Cell Signaling Technology, Danvers, MA, USA, #3662), AKT (1:1000, Abcam, Cambridge, MA, USA), ab38449), P-AKT (Ser473) (1:1000, Wanleibio, Shenyang, China, WLP001a), Insulin Receptor (INSR) (1:500, Wanleibio, Shenyang, China, WL02857), and GAPDH (1:2000, Cell Signaling Technology, Danvers, MA, USA, #5174S). The washed membranes were incubated with the corresponding secondary antibodies for 1–2 h. Finally, the signal was detected with bio rad Chemidoc Touch imager (Bio-Rad Chemidoc Touch, Hercules, CA, USA) using enhanced chemiluminescence kits (ECL, vazyme, Nanjing, China).