Phospho perk thr980

For evaluation of the protein level, cell pellets were washed in PBS and lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA), including protease/phosphatase inhibitors, vortexed, and incubated for 20 minutes on ice. Cell lysates were then centrifuged at 12,000 rpm for 15 min at 4°C. The supernatants were collected, and proteins were quantified with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). For immunoblot analyses, 20 μg of protein lysates per sample were denatured in 4X Loading dye and boiled using a heating block at 95 degrees for 10 minutes before loading to SDS gradient gels 4%−20% (Bio-Rad Criterion, 1h runs). Gels were semi-dry transferred onto PVDF, and the membranes were blocked with 3% milk in 0.1% TBST. The membraneNext, the membrane was probed with either PGC1 α antibody (Millipore Sigma, Burlington, MA), PERK antibody (Novus Biologicals, Centennial, CO), Phospho-IRE1 α (Ser724) antibody (Invitrogen, Waltham, MA), MFN2, OPA1, Phospho-PERK (Thr980), IRE1 α, LC3A/B antibody (Cell Signaling Technology, Danvers, MA), or β-Actin antibody (Santa Cruz Biotechnology, Inc, Dallas, TX) overnight at 4°C followed by one-hour incubation with HRP-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA) and using a Clarity Western ECL Substrate (Bio-Rad, Hercules, CA).