Hspa5

Cells were lysed using buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM EDTA pH8.0, 1 mM EGTA pH8.0, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100) containing the protease inhibitor mixture (Sigma-Aldrich). After sonication, the lysates were centrifuged at 15,400 × g for 5 min at 4°C. Protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA), with albumin as standards. Total proteins (20 µg) were electrophoresed on 10%−12% SDS-polyacrylamide gel and transferred onto nitrocellulose membrane (Schleicher and Schuell, Dassel, Germany) by reverse electrophoresis. After being blocked, the membrane was stained with PGC-1α, SOD2, NRF2, HSPA5, NFYA (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CYCS (1:1000; BioVision, Milpitas, CA, USA), GCLC (1:1000; Abcam, Cambridge, MA, USA), NQO1 (1:1000; Sigma-Aldrich), or GAPDH (1:1000; MDBio Inc., Taipei, Taiwan) primary antibody at 4°C overnight. The immune complexes were detected using horseradish peroxidase-conjugated goat antimouse or goat antirabbit IgG antibody (1:10000; GeneTex, Irvive, CA, USA) and chemiluminescent substrate (Millipore, Billerica, MA, USA).

Triton or digitonin-lysed samples as well as differential centrifugation samples were substituted with sample buffer (8.5% glycerin, 2% SDS, 6.25% Tris/HCl, pH 6.8, 20 mm DTT, 0.013% bromphenol blue) but not heat denatured. As a reducing agent tris(2-carboxyethyl)phosphine (Thermo Scientific) was used as described (5 (link)). The protein amount was determined by Bradford protein assay. Proteins were separated by SDS-PAGE, substituted to Western blot, and detected by primary antibodies. Infrared fluorescent-labeled secondary antibodies were used and visualized in the Odyssey system (Licor, Bad Homburg, Germany).
NOX4 was detected by an anti-NOX4 antibody (1:2,000) reported by Anilkumar et al. (24 (link)). Primary antibody against NOX1 (Mox1, number sc-5821, 1:500), NOX5 (number sc-67006, 1:500), β-Tubulin (number sc-9104, 1:500), and HSPA5 (number sc-1051, 1:1,000) were purchased from Santa Cruz. Anti-calnexin (number MAB3126, 1:2,000) was obtained from Merck Millipore; Na/K-ATPase (number α6F, 1:2,000) from Hybridoma Bank, University of Iowa; Golgin (number A21270, 1:1,000) from Invitrogen; GAPDH (number G8795, 1:10,000) and β-Actin (number A1978, 1:2,000) from Sigma; and GFP from Roche (number 11814460001, 1:2,000). Antisera for mitochondrial proteins (ATP synthase subunit α or subunit β, Complex III) were used as described previously (26 (link)).