Ms304

Tissues were collected immediately following euthanasia and flash frozen in liquid nitrogen. Homogenates were prepared by Polytron homogenization in RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails (Sigma, St. Louis, MO). Protein content was quantified by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). 15–25 μg of protein was run on a 10% SDS-PAGE gel (Bio-Rad) and transferred to a PVDF membrane (Millipore, Billerica, MA). Membranes were incubated overnight with antibodies to NAMPT (A300-372; Bethyl, Montgomery, TX), complex III (MS304, MitoSciences, Eugene, OR), and α-tubulin (ab7291, Abcam) and then probed with IRDye 680 goat anti-mouse IgG or IRDye 800CW goat anti-rabbit IgG (926-32220 and 92632211, respectively; LI-COR, Lincoln, NE). Bands were visualized using an Odyssey Digital Infrared Imaging System (LI-COR) and quantified using Odyssey Application Software version 3.0 (LI-COR).

Tissues or cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 12 mM 464 deoxycholic acid, 0.5% Nonidet P-40, and protease and phosphatase inhibitors). Ten μg proteins were loaded on SDS-PAGE and subjected to immunoblotting. Nitrocellulose membranes were incubated with anti-Ampk-p172 (#2531, Cell Signaling Technology), anti-Ucp1 (ab23841, abcam), anti-vDAC (sc-32063, Santa Cruz Biotechnology), anti-Hsp60 (sc-13966, Santa Cruz Biotechnology), anti-Ampk (sc-25792, Santa Cruz Biotechnology), anti-Hsl (#4107, Cell Signaling Technology), Pc (sc-271493, Santa Cruz Biotechnology), anti-Tom20 (sc-11415, Santa Cruz Biotechnolog), anti- Complex I ndufb8 (ms-105, MitoSciences), anti-Complex III Core2 subunit (ms-304, MitoSciences), anti-Vinculin (ab-18058, Abcam), and anti-Pdhβ (gtx-119625, GeneTex) primary antibodies, at 1:1000 dilution. Successively, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were detected with a FluorChem FC3 System (Protein-Simple, San Jose, CA, USA) after incubation of the membranes with ECL Selected Western Blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA). Densitometric analyses of the immunoreactive bands were performed with the FluorChem FC3 Analysis Software.