Western Blotting of OXPHOS and HO-1

Western blotting was performed by a modification of Hara’s method and Ota’s method^{5 (link),29 (link)}. In particular, total protein extracts (2 × 10⁴ cells/lane) were separated by polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes using the Trans-Blot^® Turbo Transfer system (Bio Rad Laboratories, USA), and incubated with antibodies. Immuno-labelled proteins were detected using a chemiluminescence kit (Immuno Star LD (FUJIFILM Wako Pure Chemical, Japan)) and a lumino-image analyser (ChemiDoc MP system (Bio Rad Laboratories, USA)). The primary antibodies used were Total OXPHOS Rodent WB Antibody Cocktail (abcam, UK) and rabbit anti-human HO-1 antibody (kindly provided by Dr. Shigeru Taketani)^{30 (link),31 (link)}. The secondary antibodies used were anti-rabbit IgG HRP-Linked Whole Ab Donkey (GE Healthcare, USA) for OXPHOS proteins and anti-mouse IgG HRP-Linked Whole Ab Sheep (GE Healthcare, USA) for HO-1. Beta-actin was used as an internal control. Anti-beta-actin antibody (ab8227) (abcam, UK) was used as primary antibody and anti-rabbit IgG HRP-Linked Whole Ab Donkey (GE Healthcare, USA) was used as secondary antibody.