Western blotting was done as previously described (Stice et al., 2011 (link)). The membrane was stained with Ponceau after transfer to verify the proteins were equally loaded. An internal control was used to allow comparison among different membranes. Values were normalized to the internal control to correct for variations amongst blots as previously described (Voss et al., 2003 (link)). The same mitochondrial samples were analyzed for ATP-synthase (ab109867, Abcam, Cambridge, MA), NADH dehydrogenase flavoprotein 1 (NDUFV1) (ab55535, Abcam, Cambridge, MA), Tripartite motif-containing protein 72 (TRIM72) (ab68061, Abcam, Cambridge, MA), Heat shock cognate 70 (HSC 70) (PA5–29241, Thermo Scientific, Rockford, I.L.), and Voltage- dependent anion channel-1 (VDAC-1) (ab15895, Abcam, Cambridge, MA) with 1:1000 dilution, and NADH dehydrogenase 1 alpha subcomplex subunit 5 (NDUFA5) (ab119308, Abcam, Cambridge, MA) with 1:100 dilution. Anti-mouse secondary antibody and anti-rabbit secondary antibody (respectively NA931V, NA934V GE Healthcare, Pittsburgh, PA) were used at a 1:1000 dilution.
After washing, the membrane was developed with a chemiluminescent system and analyzed as previously described (Stice, Chen, 2011 (link)).
After washing, the membrane was developed with a chemiluminescent system and analyzed as previously described (Stice, Chen, 2011 (link)).