Pgc 1α

The total protein was extracted by lysate. A BCA kit was used to quantify protein concentration; then, protein was transferred to the PVDF membrane, and after being resolved by SDS-PAGE (Millipore, Carrigtwohill, Ireland), DRP1 (1:500; Affinity Biosciences, OH, USA), MFN2 (1:1000; Affinity Biosciences, OH, USA), Adiponectin (APN, 1:1000; Zen BioScience, Chengdu, China), AMPK (1:1000; Affinity Biosciences, OH, USA), p-AMPK (1:1000; Affinity Biosciences, OH, USA), SIRT1 (1:2000; Boster, Wuhan, China), PGC-1α (1:1000; Zen BioScience, Chengdu, China), NRF1 (1:1000; Zen BioScience, Chengdu, China), TFAM (1:2000; Affinity Biosciences, Chicago, IL, USA) and β-actin (1:1000; internal control, Boster, Wuhan, China) were incubated together with the membrane in TBST overnight. A TBST wash and 2 h incubation with secondary antibodies followed. Lastly, protein signals were detected with an ECL kit (Boster, Wuhan, China).

Antibodies from Affinity Biosciences: p-Smad1/5/8 (1:1000, #AF8313), Smad1/5/8 (1:1000, #AF0614), PPARγ (1:1000, #bs-4590R), UCP1 (1:1000, #72298). Antibodies from Beyotime: HA tag (1:1000, #AH158), Flag tag (1:1000, #AF519). Antibodies from Sangon Biotech: p-ACC (1:1000, #D155180), ACC (1:1000, #D155300). Antibodies from Abcam: BMP8A (1:1000, #154373). Antibodies from ZenBio: p-AMPK (1:1000, #R26252), AMPK (1:1000, #380431), PGC-1α (1:1000, #381615). Antibodies from CWBIO: goat anti-Rabbit IgG HRP secondary antibody (1:4000, #CW0103S), goat anti-Mouse IgG HRP Conjugated (1:4000, # CW0102). Antibodies from Bioss: p38 MAPK (1:1000, #bs-0637R), p-p38 MAPK (1:1000, #bs-2210R), β-actin (1:2000, #bs-0061R), p-JNK (1:1000, #bs-1640R), JNK (1:1000, #bs-2592R), C/EBPα (1:1000, #AF6333), p-Smad2/3 (1:1000, #AF3367), Smad2/3 (1:1000, #AF6367), p-ERK1/2 (1:1000, #AF1015), ERK1/2 (1:1000, #AF0155), p-p65 (1:1000, #AF2006), p65 (1:1000, #AF5006), p-IKKα/β (1:1000, #AF3013), IKKα/β (1:1000, # AF6014).

The specific process followed the previous article.
³⁵ (link) Briefly, proteins were isolated from cells by using RIPA lysis buffer and separated by 10% SDS‐polyacrylamide gel electrophoresis. Then they are transferred to PVDF membranes. Thereafter, the membranes were incubated with primary antibodies against β‐Actin (sc‐47778, Santa Cruz Biotechnology), Mfn1 (No. 509880, ZEN BIO), Mfn2 (No. 340604, ZEN BIO), Opa1 (No. 382025, ZEN BIO), phospho‐AMPKα1 (No. R26252, ZEN BIO), AMPKα1 (No. 380431, ZEN BIO), PGC1α (No. 381615, ZEN BIO), SIRT1 (No. R25721, ZEN BIO), Smad3 (#ab28379, Abcam), phospho‐Smad3 (No. R22919, ZEN BIO), Smad4 (#ab40759, Abcam), Drp1 (No. 221099, ZEN BIO) and Fis1 (No. R26001, ZEN BIO) at 4°C overnight, and the secondary antibodies against rabbit (sc‐2357, Santa Cruz Biotechnology) and mouse (sc‐516102, Santa Cruz Biotechnology) were incubated for 1 h at room temperature. Immobilon® Western kit (P90719, Millipore, Massachusetts, USA) was used to visualize the protein bands.