Western Blot Analysis of Respiratory Proteins

For western analysis of respiratory proteins, WCEs were prepared by trichloroacetic acid extraction as previously described (Reid and Schatz, 1982 (link)) and immunoblot analysis was conducted as described previously (Nanda et al., 2009 (link)). After electroblotting to polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010), membranes were probed with antibodies against Aco1, Atp20, Cox14, Pet10 (a kind gift from Dr. Nikolaus Pfanner), Idh1 (Abnova, PAB19472), and GAPDH (Proteintech, 60004). Secondary antibodies employed were HRP-conjugated anti-rabbit (GE, NA9340V), anti-mouse IgG (GE, NA931V), and anti-goat IgG (Abnova, PAB29101). Detection was performed using enhanced chemiluminescence (ECL, Cytiva, RPN2109) and the Azure 200 gel imaging biosystem.