Phospho atm

Cells were lysed in RIPA Buffer (Boston BioProducts #BP-115), supplemented with protease inhibitor cocktail tablets (Roche, complete mini, #11 836 153 001), and 5 mM NaF. Protein was quantified via Bio-RAD Protein Assay Dye Reagent Concentrate (BIO-RAD, #500-0006) on the Beckman Coulter DU640 Spectrophotometer. Proteins were separated by PAGE and electo-transferred to PVDF membranes (Pall Corporation, BioTrace PVDF 0.45 um, P/N 66543). Membranes were blocked in 5% milk tris-buffered saline with tween 20 (0.1%; TBS-T). Primary antibodies were incubated overnight on a rocker in 5% bovine serum albumin in TBS-T at 1:1000 at 4°C. Membranes were washed the following day 3×, 5′, TBS-T, and incubated with either mouse or rabbit horseradish peroxidase secondary (Cell Signaling #7076S and #7074) for 2 hours room temperature. Proteins were detected via film following the Thermo Scientific's SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, #34087) or Thermo Scientific's SuperSignal West Femto Maximum Sensitivity Substrate (#34095) protocol. The following antibodies were purchased from Cell Signaling Technology: B-Actin (#3700), BiP (#3177), CHOP (#2895), ATF-4 (#11815), Rad51 (#8875), Phospho-Histone H2A.X (#2577), H2A.X (#2595), ATM (#2873), Phospho-ATM (#13050), CHK2 (#6334), Phospho-Chk2 (#2661).

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.

Total cellular protein lysates were prepared with Pierce lysis buffer (Pierce, Rockford, IL, USA) and quantified by the Bradford method. Equal amounts of total protein (20 µg) were loaded onto 10% SDS‐PAGE gels and electrophoresed at 100 V for 1 hour. Then, the separated proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA) at 80 V for 2 hours. The membranes were blocked in 5% non‐fat milk in 1× TBST and then incubated with primary antibodies against EGFR (1:500; Santa Cruz, USA), PERK, IRE1α, ATF 6, (1:1000; Abcam), phospho‐eIF2α, GRP78, GRP94, PDI, ERO1‐Lα, CHOP, phospho‐ATM, DNA‐PK, LC3B, Atg3, cleaved caspase 3, cleaved PARP, and β‐actin (1:1000; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight. After washing with 1× TBST, the membranes were incubated with secondary anti‐mouse or anti‐rabbit IgG HRP‐linked antibodies (1:5000; Cell Signaling Technology) at room temperature for 2 hours. The blots were developed with Target LumiGLO (Cell Signaling Technology) and photographed with DNR BioImaging System (DNR, Israel).