Pageruler prestained marker

Proteins were analyzed by SDS–PAGE (12.5% acrylamide) in reducing conditions with the PageRuler prestained marker (Thermo Fisher Scientific), 5 µl/lane. The gels were stained with colloidal Coomassie blue, de-stained with the deionized water until clear background, scanned using the flatbed scanner in the transparent mode, saved as 16-bit grayscale images. The images were analyzed using the TotalLab TL120 software (Nonlinear Dynamics, Newcastle upon Tyne, UK).

SDS-PAGE was performed with the 12.5% acrylamide in the separating gel, in reducing conditions, if not stated otherwise, with the PageRuler prestained marker, 5 μl/lane (ThermoFisher Scientific). Gels were stained by the colloidal Coomassie blue according to [27 (link)], scanned by the conventional flatbed scanner in the transparent mode as 16-bit grayscale images and analyzed by the TotalLab TL120 gel densitometry software (Nonlinear Dynamics, UK).

Fetal bovine serum (FBS) (10270-106) was purchased from Gibco (NY, USA); high-glucose DMEM (SH30022.01B) from HyClone (USA); the CCK-8 assay kit from Dojindo (Japan); the TUNEL assay kit (11684795910) from Roche (Switzerland); 0.25% trypsin-EDTA (C0201), penicillin-streptomycin liquid (C0222), RIPA lysis solution (P0013B), PMSF (ST506), DAPI staining solution (C1006), and the BCA Protein Assay kit (P0012) from Beyotime Biotechnology (Shanghai, China); the SDS-PAGE Preparation kit (C831100-0200) and EZ-10 Total RNA Mini-Preps Kit (B618583) from Shenggong Biotech Inc. (Shanghai, China); SDS (S8010), glycine (G8200), and Tris (T8060) from Beijing Suo Laibao Technology Co., Ltd. (Beijing, China); PageRuler prestained marker (26616) from Thermo Fisher Scientific (MA, USA); anti-Beclin-1 (ab20761) and anti-ATG-5 (ab108327) antibodies from Abcam (Cambridge, UK); anti-p62 (88588S), anti-LC3 (3868S), anti-mTOR (2983T), and anti-p-mTOR (5536T) antibodies from Cell Signaling Technology (Danvers, MA, USA); ABScript II RT Master Mix for qPCR with gDNA Remover (RK20403) from ABclonal (Wuhan, China); TB Green™ Premix Ex Taq™ II (Tli RNase H Plus) (RR820A) from TaKaRa (Dalian, China); rapamycin (HY-10219) from MedChemExpress (MCE, NJ, USA); and MHY1485 (S7811) from Selleck (Houston, TX, USA).