Protein Extraction and Western Blot Analysis

Cells grown to confluence on 6-well culture plates (VWR) were harvested after the specified treatment period and washed with PBS. Protein was extracted from the cells using mammalian protein extraction reagent with protease inhibitor cocktail (Pierce Biotechnology, Rockford, IL), and quantified with a protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were resolved on 8–16% Tris-HCL polyacrylamide gels (Pierce Biotechnology, Rockford, IL) and transferred to PVDF blotting membranes (Millipore, Billerica, MA). Membranes were probed with rabbit polyclonal anti-gamma glutamyl cysteine ligase, catalytic subunit (GCLC) (Abcam, Cambridge, MA), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, MA), mouse anti-CHOP (Pierce Biotechnology, IL), rabbit anti-GRP78 (Sigma Aldrich, St. Louis, MO), rabbit anti-COX IV (Cell Signaling Technology, MA), rabbit polyclonal anti-GRX2 (GeneTex, Irvine, CA) and rabbit anti-β-Tubulin (Cell Signaling Technology, MA) overnight at 4°C. After incubation with corresponding secondary antibody tagged with horseradish peroxidase, signals were detected using an ECL chemiluminescence system (Pierce Biotechnology, IL). Membranes were then stripped and reprobed with monoclonal anti-GAPDH (Millipore, Billerica, MA). Protein band intensity was measured by Image Studio Software (Li-Cor, Lincoln, NE) [30 (link)].