Mouse anti pgc1α

Cells were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate [SDS]) containing protease inhibitors (1 mM phenyl-methyl sulphonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin). Cell lysates were spun at 13,000 rpm for 30 min to remove debris, and protein quantification was performed using the Bio-Rad DC Protein Assay kit (RRID:SCR_008426). Total protein (40 μg/well) was fractionated by 10% SDS–PAGE. Fractionated proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in 5% non-fat dry milk in PBS containing 0.1% Tween 20 (PBST). Blots were incubated overnight at 4°C with primary antibodies: ALDH1A1, 1:1000 (Santa Cruz Cat# Sc 374149), mouse anti-PGC1α, 1:1000 (Santa Cruz Cat# 518025) and Smad4, 1:1000 (RRID:AB_627905). β-actin was used as a loading control for protein normalization. The membranes were then washed in PBST, probed with the appropriate secondary antibodies, incubated for an hour at room temperature, and then washed with PBST. Signals were detected with the ECL chemiluminescence kit (Thermo Scientific).