Isolation and Analysis of Mitochondrial Fractions

Cells were harvested following 0.25% trypsin dissociation, quenched in full medium, and washed in PBS prior to cell counting using a Cellometer Auto 2000 Cell Viability Counter (Nexcelom Bioscience, Lawrence, MA). Batches of equal cell count were aliquoted (2-4 million per experiment), and cell viability was confirmed >90% through AOPI double staining with ViaStain (Cat. #CS2-106, Nexcelom Bioscience). Mitochondria isolation, protein quantification, and enrichment of soluble and insoluble mitochondrial fractions were performed as previously described^{5 (link)} following manual lysis using a Dounce homogenizer on ice (40 strokes per cell type in 0.8 mL SM buffer consisting of 50 mM Tris-HCl pH 7.4, 0.25 M sucrose, 2 mM EDTA, and 1% BSA). Equal volumes of mitochondrial fractions were examined by western blot (Cat. #4568123 and #1704156, Bio-Rad Laboratories) and probed with 1:2,000 anti-HAX1 antibody (Cat. #ab137613 and #ab6721, Abcam) in 1% milk/TBST overnight after blocking in 5% milk/TBST.