Ab54481

In this study, the western blot assay was performed as our previously described [34 (link), 35 (link)]. Briefly, the protein sample (20 µg/lane) was separated using 8% or 12% SDS-PAGE gel and then transferred to nitrocellulose membranes. The membrane was blocked using 5% bovine serum albumin, then incubated with primary antibodies overnight at 4 ˚C. In the present study, the primary antibodies used were: anti-COX7A1 (1:3000; Abcam, ab123591, USA), anti-p62 (1:3000; Abcam, ab56316, USA), anti-LC3 (1:3000; Sigma, L7543, USA), anti-TMO20 (1:1000; Santa Crus, sc-17764, USA), anti-TIM23 (1:2000; Abcam, ab230253, USA), anti-DRP1 (1:2000; Abcam, ab184247, USA), anti-MFN1 (1:1000; Proteintech, 13798-1-AP, USA), anti-VDAC (1:2000; Proteintech, 10866-1-AP, USA), anti-PINK (1:2000; Abcam, ab137361, USA), anti-PARKIN (1:2000; Abcam, ab77924, USA), anti-PGC-1α (1:3000; Abcam, ab54481, USA), anti-TFAM (1:1000; Cell Signaling, 7495, USA) and anti-GAPDH (1:3000; Santa Cruz, sc-47724, USA). After incubated with HRP-labeled secondary antibodies, the protein bands were visualized using an enhanced chemiluminescence kit (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, USA) as well as ChemiDoc Imagers (Bio-Rad Laboratories, USA). Full-length western blots can be found in Supplemental Materials.

Cells were lysed by lysis buffer (10 mM Tris, 1 m MEDTA,1% Triton X-100, 1 mM Na₃VO₄, 20 μg/mL aprotinin, 20 μg/mL leupeptin, 1 mM dithiothreitol, and 50 μg/mL phenylmethylsulfonyl fluoride). Cell lysates were subjected to 10% SDS-PAGE and proteins were transferred to the PVDF membrane by an electro-transfer unit. Membranes were probed with primary antibodies overnight at 4  °C. The primary antibodies included anti-Bcl-2 (ab692, Abcam; 1:2000), anti-caspase-3 (ab32351; 1:2000), anti-Bax (#89477, cell signaling technology, Beverly, MA, USA; 1:2000), anti-PGC1-α (ab54481; 1:2000), anti-NRF1 (ab34682; 1:2000), anti-PKC (ab181558; 1:2000), anti-HMGB1 (ab18256; 1:2000), anti-RAGE (ab216329; 1:2000), and anti-NFκB (ab16502; 1:2000). Following incubation of primary antibodies, the membranes were rinsed three times and incubated with corresponding secondary antibodies. The immunoreactive bands were developed using a Western-Ready™ ECL Substrate Plus Kit (426316, BioLegend, San Diego, CA, USA) and detected by MultiGel-21^® image system. β-actin was used as an internal control.