Las4000 imaging system

Cells were lysed with 1 % NP40 lysis buffer (1 % NP40, 300 mM NaCl, 0.1 mM EDTA, 50 mM Tris [pH 7.5]) supplemented with protease inhibitor cocktail (Roche) and resolved by NuPAGE (Invitrogen) gels and transferred onto nitrocellulose membrane followed by antibody detection using enhanced chemiluminescence method (Western Lightening, Perkin Elmer). Some blots were developed with LAS-4000 Imaging system (GE Healthcare Life Sciences). For immunoprecipitation, cells were lysed with 150 mM NaCl buffer (1 % NP40, 150 mM NaCl, 0.1 mM EDTA, 50 mM Tris [pH 7.5]), and the lysates were incubated with anti-Flag agarose (Sigma) or anti-HA agarose (Sigma) followed by three times washing with 150 mM NaCl buffer. For cellular fractionation, cells were incubated with low salt permeabilization buffer (10 mM HEPES [pH 7.4], 10 mM KCl, 0.05% NP-40) on ice for 20 min. Following centrifugation, pellet was boiled in boiling lysis buffer (50 mM Tris [pH 6.8], 2 % SDS, 850 mM β-mercaptoethanol). Subcellular fractionation of HeLa cells using 100 mM NaCl concentration was performed as previously described (Kim et al., 2012 (link)).

Antibodies against MsrB1, Nrf2, heme oxygenase-1 (HO-1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as previously described [23 (link),24 (link)]. Antibodies against histone 1 and HSP90 were purchased from Santa Cruz Biotechnology and Cell Signaling Technology, respectively. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) using a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and transferred onto a polyvinylidene difluoride membrane. Membranes were subsequently probed with primary antibodies, followed by the addition of horseradish peroxidase-conjugated secondary antibodies. GAPDH expression was used as the protein loading control. Quantitative analysis of blot signals was performed using the LAS-4000 imaging system (GE Healthcare Life Sciences) and ImageJ software (open source).