Western Blot Analysis of Protein Levels

To evaluate protein levels, cell lysates were separated by SDS-PAGE and analyzed by Western blot as described^{10 (link)}. Briefly, cell pellets or snap-frozen tissues were homogenized in a lysis buffer containing 10 mM Tris-HCl, pH 6.8–7.5, 2 mM EDTA, 0.5% SDS, and freshly added 2-mercaptoethanol. Tissue homogenates were centrifuged at 12,000 g for 15 min at 4 °C, and the supernatants were collected. The total protein extracts were aliquoted to determine protein concentration using a protein assay dye reagent concentrate (Bio-Rad, CA, USA). About 100 μg total protein was separated by SDS-PAGE and then transferred to nitrocellulose membrane. The membranes were probed with the following antibodies against p22phox (1:500; sc-20781, Santa Cruz), GCLC (1:600; sc-28965, Santa Cruz), GR (1:500; sc-13324, Santa Cruz), and β-actin (1:10000, Sigma-Aldrich), washed with PBST and then probed with their respective secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. Autoradiography or the Odyssey Imaging System (LiCor Biosciences, Lincoln, NE) was used to visualize protein bands. Stripping buffer (Thermo, MA, USA) was used for sequential blotting and probing with other antibodies. ImageJ densitometry software was used to quantify individual bands.