Ab55435

Total protein, nucleoprotein and cytoplasmic protein of MRC-5 cells were extracted using Radio Immunoprecipitation Assay Buffer (Beyotime, Shanghai, China) and EpiQuik Nuclear Extraction Kit (Epigentek, Farmingdale NY, USA), respectively. Protein samples were quantified by Bio-Rad Protein Assay (Bio-Rad, Hercules CA, USA) and then loaded to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands on the gel were then transferred to polyvinylidene fluoride membranes, which were then blocked in 5% skim milk for 4 h at room temperature. The membranes were incubated in Tris-buffered saline and Tween 20 (TBST) containing the specific primary antibodies against NRF2, KEAP1, NQO1, GCLC, HO1 and GAPDH (ab89443, ab150654, ab28947, ab55435, ab13248 and ab8245; Abcam, Cambridge, UK) overnight at 4 °C. After washed in TBST for five times, the membranes were incubated in horse radish peroxidase-conjugated secondary antibodies (ab6708, Abcam) for 2 h at room temperature, and then washed again in TBST for five times. Protein signals were developed using ECL Plus Western Blotting Substrate (Pierce, Carlsbad CA, USA).

DEX, polybrene, puromycin, JC-1 dye, MG-132, cycloheximide (CHX), and 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) dye were obtained from Sigma-Aldrich Chemicals (St. Louis, MO). The following antibodies for p44/42 MAPK (Erk1/2, #9102), HO1 (#70081), NQO1 (#3187), Nrf2 (#12721), Keap1 (#8047), α-Tubulin (#2125), and Lamin B1 (#13435) as well as cleaved-poly (ADP-ribose) polymerase (PARP, #5625), cleaved-caspase-3 (#9664) were purchased from Cell Signaling Tech (Shanghai, China). The anti-GCLC antibody (ab55435) and the anti-adenine nucleotide translocase 1 (ANT-1) antibody (ab102032) were provided by Abcam (Shanghai, China). The following antibodies for PGK1 (sc-130335), cyclophilin-D (CyPD, sc-137136), and VDAC1 (sc-390996) were purchased from Santa Cruz Biotech Co. (Santa Cruz, CA). The reagents for cell culturing, including fetal bovine serum (FBS), DMEM, penicillin and streptomycin were obtained from Gibco-BRL Co. (Grand Island, NY). TRIzol and other RNA assay agents, Annexin V, propidium iodide (PI) and cell transfection reagents (Lipofectamine 2000 and others) were from Invitrogen Thermo-Fisher (Shanghai, China). mRNA primers were purchased from Genechem Co. (Shanghai, China). All viral constructs and sequences were also designed and provided by Genechem Co.