Anti gpx 1

RAW 264.7 cell lysates were made using a standard procedure, mixed with sample buffer consisting of Tris-HCl (250 mM, pH 6.8), 0.5 M dithiothreitol (DTT), glycerol (50%), bromophenol blue (0.5%,), SDS (10%), and 2-mercaptoethanol (5%), and denatured at 100 °C for 5 min. A nuclear/cytosolic fractionation kit (Cell Biolabs, Inc., San Diego, CA, USA) was employed for nuclear protein extraction. SDS-PAGE (10%) was employed to separate the sample proteins (20 μg), and the membranes were incubated overnight with the primary antibody in skim milk (5%, w/v) after electrotransfer on nitrocellulose membranes (Whatman, Dassel, Germany). Primary antibodies (1:1000), including anti-SOD1 (sc-101523), anti-CAT (sc-515782), anti-GPx-1 (sc-133152), anti-HO-1 (sc-136256), anti-Nrf2 (sc-81342), β-actin (sc-47778), and lamin B (sc-374015) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), were applied. Anti-Mouse IgG-HRP (Santa Cruz) was employed as a secondary antibody. An ECL solution method was used to identify the antigen–antibody reaction. The density of the protein bands was normalized using the same samples on the β-actin.

Cells were harvested in ice-cold PBS and lysed in RIPA buffer (Cell signaling) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche) for 20 min on ice. Fifty millimolar of N-Ethylmaleimide (NEM; Sigma) was supplemented for s-glutathionylation preparations. After centrifugation at 15,000×g for 15 min at 4 °C, total protein in whole-cell extracts was quantified using Rotiquant (Carl Roth). Forty micrograms of protein were resolved by SDS-PAGE (Invitrogen) and blotted on PVDF membranes (Invitrogen). The membranes were probed with anti-GSTP1, anti-xCT, anti-catalase, anti-SOD1, anti-GPX1, anti-γGCS, or anti-β actin (Santa Cruz) primary antibodies followed by secondary horse-radish peroxidase (HRP) coupled antibodies (Santa Cruz). Signals were acquired in a chemiluminescence detection system (Applied Biosystems) in a linear dynamic range.