Criterion apparatus

Antibodies used were: anti-HA (Abcam, 1∶2000), anti-human SIRT3 (Epitomics, 1∶500), anti-human SIRT4 (Antibodies-Online, 1∶500), anti-human SIRT5 (Abcam, 1∶5000), anti-cytochrome-C (Pierce, 1∶500) anti-very long-chain acyl-CoA dehydrogenase (1∶1000; gift of Dr. Jerry Vockley), and respiratory chain antibody cocktail (1∶1000; Mitosciences, Eugene, OR). Cells were lysed in RIPA buffer and the homogenates were cleared by centrifugation and analyzed for protein concentration in triplicate using the Bradford method (Bio-Rad Hercules, CA). Lysates were electrophoresed and transferred to nitrocellulose membranes using the Bio-Rad Criterion apparatus. For western blotting of cell fractions, cell pellets were gently dispersed in 250 mM sucrose, 1 mM EDTA, 10 mM Tris, pH 7.4. The cell suspensions were lysed mechanically by 20 passes through a cell homogenizer (Isobiotech, Heidelberg, Germany) using 10 µM clearance. Unbroken cells and nuclei were removed and discarded by centrifugation at 1,000×g for 10 minutes. Mitochondria were collected by centrifuging the supernatant at 12,000×g for 15 minutes. The supernatant was taken as the cytosolic fraction and the pellet as the mitochondrial fraction.

Protein content was measured from total protein extracts by western immunoblotting using the Criterion apparatus and 12.5% SDS-polyacrylamide gels (all purchased from Bio-Rad, Hercules, CA) and adjusted to either GAPDH (AB9484; AbCam, Cambridge, MA) or total protein assessed by Ponceau S stain (Sigma, St. Louis, MO). The antibody for PLIN3 was purchased from Novus Biologicals (Cat no. NB110-40764, Littleton, CO). The antibodies for GBF1 (AB86071), ATGL (AB109251), and ARFRP1 (AB108199) were purchased from AbCam (Cambridge, MA). The antibody for ARF1 was purchased from Epitomics (Cat no. 1635-1; Burlingame, CA).