Rabbit anti cytochrome c antibody

Lung tissues were washed twice with ice-cold PBS and lysed in lysis buffer [10mM tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 1mM phenylmethylsulfonyl fluoride, and leupeptin and aprotinin (10 mg/ml each)]. After centrifugation at 12,000g for 15 min, the supernatants were collected and their protein concentrations were measured using a BCA protein assay kit (HyClone Pierce). The tissue homogenates (50 μg of protein) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The blots were then washed with tris-buffered saline with Tween 20 (TBST), blocked with 5% milk powder in TBST buffer for 1 hour, and incubated with the appropriate primary antibodies at appropriate dilutions. The blotted membranes were probed with the rabbit anti-irisin antibody (1:500; Phoenix Pharmaceuticals), the rabbit anti-UCP2 antibody (1:500; Sigma-Aldrich), rabbit anti-active caspase 9 antibody (1:500; Abcam), and rabbit anti–cytochrome c antibody (1:500, Abcam) at 4°C overnight. Then, the membranes were washed and primary antibodies were detected with fluorescent-labeled goat anti-rabbit IgG (1:15,000; IRDye, LI-COR), and the bands were visualized by Odyssey Western Blot Detection System (LI-COR). The amount of protein transferred onto the membranes was verified by immunoblotting for glyceraldehyde-3-phosphate dehydrogenase.