Mitochondrial Stress Response Evaluation

MSCs exposed to normoxia (5%) or hyperoxia (85%) for 24 h were lysed in RIPA buffer (Alfa Aesar. Cat. No. J62885) and the lysate was centrifuged at 6,000 g for 10 min at 4ºC, then the supernatant was mixed with 4 × Laemmli sample buffer for western blotting according to standard procedures. 30 to 60 µg of total protein from the samples were separated on 4–20% Criterion TGX Gels (Bio-Rad, Cat. No. 5678093), transferred to PVDF membranes (Trans-Blot Turbo System, Bio-Rad) and probed with antibodies against PINK1 (ab23707), Aconitase (ab181153),TOM20 (ab56783, Abcam, MA) and B-Actin (Cell Signaling Technology, 5125S) as loading control^{61 (link)}. Membranes were developed using Clarity ECL Substrate (Bio-Rad) and visualized with ChemiDoc imaging System. Densitometry analysis of band intensities was performed using ImageJ software.