Surepage gel

After removal of culture medium, neurons were washed three times with PBS, and immediately lysed using 1 ml of RIPA (Sigma, America) containing protease and phosphatase inhibitor cocktails (Roche, America) to obtain total proteins. BCA kit (Beyotime, China) was used to quantify proteins in the samples. For immunoblotting, equal amounts of proteins were separated by 8–16% SurePageTM Gels (GenScript, USA), transferred to polyvinylidene difluoride membrane (Roche, America) and then blocked for 1 h using 5% BSA diluted in TBS-T. Membranes containing proteins were incubated with LC3, Beclin1, p62, ULK1, PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, p70S6, p-p70S6, 4ebp1, p-4ebp1, β-actin (1:2,000; all from CST, America) primary antibodies overnight. Membranes were then incubated with the corresponding secondary HRP-coupled anti-rabbit antibody (1:5,000; CST, America) for 2 h. ECL detection system (Tanon, China) was used to visualize bands. For quantitative analysis, the bands were analyzed using ImageJ software.

The significance of differences between mean values of control and test samples was compared using Student's t test in the open‐source software suite “R” (http://cran.r-project.org/). Differences with p < .05 were regarded as obvious, p < .01 as significant, and p < .001 as very significant. The SDS‐PAGE was run using the commercially purchased SurePageTM Gels (GenScript). The protein mass spectrometry was performed using the Orbitrap Fusion Lumos Tribrid Mass Spectrometer (LC‐MS) (Thermo Fisher), and the methods could be referred to references (Espadas, Borras, Chiva, & Sabido, 2017; Li, Zhou, Xiao, Li, & Tian, 2018).

Cells were digested with low-concentration trypsin and collected into tubes. We used a Nuclear and Cytoplasm Protein Extraction Kit (Beyotime) to separate nuclear and cytoplasm proteins. Cells were lysed in an ice cold radio immunoprecipitation assay (RIPA) lysis buffer with 1 mM phenylmethyl sulfonyl fluoride (Sigma-Aldrich), which was used for cell protein extraction. Protein concentration was determined using a BCA KIT (Beyotime). Proteins were separated by 4%–12% SurePAGE gels (GenScript, Nanjing, China), transferred to a nitrocellulose membrane (Pall, Mexico), and then detected using antibodies according to standard procedures. Primary antibodies were applied overnight at 4 °C for western blot tests, and their dilutions were as follows: NCAPG (sc-101014, Santa Cruz, 1:1000); MYHC (MF20, Developmental Studies Hybridoma Bank, 1:50); MYOD (sc-377460, Santa Cruz, 1:1000); MYOG (sc-12732, Santa Cruz, 1:1000) and β-tubulin (10094-1-AP, Proteintech, 1:2000). Finally, secondary antibodies were visualized with HRP-conjugated secondary antibodies that were applied for 1 h at room temperature. ECL western blotting detection reagent (Beyotime) was used to visualize the protein bands.