Evaporated skimmed milk

Cells were lysed in cell lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100) (Beyotime, Beijing, China) supplemented with 0.5 mM phenylmethanesulfonyl fluoride (Beyotime), and the total cellular protein concentration was determined with a BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Rockford, USA). A total of 50 μg of protein was separated on SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Membranes were then blocked with 5% evaporated skimmed milk (Bio-rad, USA) in Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.1% Tween-20 for 1 h, and probed overnight at 4 °C with the following primary antibodies: antibodies against human MGMT, phospho-ATM, phospho-CHEK1/2, phospho-H2AX, β-Catenin, active β-Catenin, phospho-GSK-3β (all 1:1000; Cell Signaling Technology, Danvers, MA, USA), antibody against Ubiquitin (Proteintech, China), antibody against ACTB (1:2000; Zsbio, Beijing, China), followed by incubation with horseradish peroxidase coupled secondary anti-mouse or anti-rabbit antibodies (Zsbio, China) for 1 h at room temperature. The protein bands were visualized using ECL-blotting detection reagents (Bio-rad, USA), and developed and fixed onto X-ray films. ACTB was served as a loading control.

Fifty μg quantity of protein was separated on SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Membranes were then blocked with 5% evaporated skimmed milk (Bio-rad, USA) in Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.1% Tween-20 for 1 h, and probed overnight at 4 °C with the following primary antibodies: antibodies against human LC3B, SQSTM1/P62, HSP60 (1:1000; Cell Signaling Technology, CST), antibody against RAB7A, RILP, EEA1, LAMP2, ACTB (1:1000, Proteintech, China), antibody against LAMP1 (1:500; Santa Cruz, USA), followed by incubation with horseradish peroxidase coupled secondary anti-mouse or anti-rabbit antibodies (Proteintech) for 1 h at room temperature. The protein bands were visualized using ECL blotting detection reagents (Bio-rad, USA), and developed and fixed onto x-ray films. ACTB was served as a loading control.