Western blotting

Harvested cells were lysed in RIPA buffer (Thermo Fisher Scientific), and then equal amounts of lysate proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA) using a Trans-Blot Turbo (Bio-Rad). After blocking with Bullet Blocking One (Nacalai Tesque, Kyoto, Japan), the membranes were incubated with primary antibodies against SQSTM1 (1:5000, {"type":"entrez-nucleotide","attrs":{"text":"BC017222","term_id":"16878015","term_text":"BC017222"}}BC017222; ProteinTech, Chicago, IL, USA), NRF2 (1:2000, SAB1303359; Sigma-Aldrich, Tokyo, Japan), HO-1 (1:2000, ab13248; Abcam), KEAP (1:5000, {"type":"entrez-nucleotide","attrs":{"text":"BC002930","term_id":"12804150","term_text":"BC002930"}}BC002930; ProteinTech), SIRT1(1:5000, {"type":"entrez-nucleotide","attrs":{"text":"BC012499","term_id":"15214729","term_text":"BC012499"}}BC012499; ProteinTech) or GAPDH (1:5000, 5A12; Fujifilm Wako Pure Chemical Corp.). Immunoreactive bands were detected using enhanced chemiluminescence (Merck Millipore, Burlington, MA, USA) after incubation with horseradish peroxidase-labeled goat anti-rabbit or anti-mouse IgG (1:5000; Vector Laboratories, Burlingame, CA, USA). Signals were detected using a C-DiGit Blot Scanner (LI-COR), and the relative band density was quantified using Image Studio DiGit software (version 5.2)³⁸.