Odyssey infrared scanner

Protein was extracted from cells by homogenization in RIPA buffer supplemented with protease inhibitor. Protein content were measured using Pierce^™ BCA Protein Assay Kit. Proteins were resolved on 4–12% bis-Tris gels and transferred 30min to nitrocellulose membrane using BioRad Trans-Blot® Turbo^™ transfer system. The blots were then incubated overnight at 4°C with primary antibody. Rabbit polyclonal antibody against mouse MitoNEET was utilized at 1:1000 as previous described (Kusminski et al., 2012 (link)). Total OXPHOS Rodent antibody cocktail was used at 1:1000 and 10ug of Rat Heart Tissue Lysate- Mitochondria extract was used as positive control in OXPHOS Western blotting. Rabbit Anti-Prohibitin antibody - Mitochondrial Marker was used at 1:1000 dilution. Monoclonal Anti-β-Actin antibody was used at 1:10 000 dilution. Primary antibodies were detected using secondary immunoglobulin Gs labeled with infrared dyes emitting at 700 nm or 800 nm. Membrane was incubated with secondary antibody for 1h at room temperature. Secondary IR-Dye 800CW Donkey antibody against rabbit was used at 1:5000. Secondary IR-Dye 680RD Donkey antibody against Mouse was used at 1:5000. Secondary IR-Dye 800CW Goat antibody against Mouse was used at 1:5000. Western blots were then visualized on a Li-Cor Odyssey infrared scanner (Li-Cor Bioscience). The scanned data were analyzed using ImageJ software

Protein extractions were done as previously described. Primary antibodies phospho- HSL (catalog no.4139; Cell Signaling Technology), tubulin (catalog no. sc-53030; Santa Cruz Biotechnology), and actin (catalog no. A4700; Sigma-Aldrich) were used at 1:1,000 dilutions and detected using a secondary immunoglobulin G labeled with infrared dyes emitting at 680 nm (catalog no. 926–68070 and 926–68076; LI-COR Bioscience) or 800 nm (catalog no. 926–32211; LI-COR Bioscience) (both at 1:10,000 dilutions) and then visualized on a LI-COR Odyssey infrared scanner (LI-COR Bioscience). The scanned data were analyzed using Odyssey version 3.0 software (LI-COR Bioscience).

PLB-985 cells differentiated for 6 days in the presence of 1.3% DMSO were resuspended in ice cold lysis buffer (20 mM Tris pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 μg/ml Pepstatin A, 200 μM PMSF, 1 μg/ml Leupeptin, and 2 μg/ml Aprotinin) and placed on ice for 10 min. Lysates were sonicated for 20 seconds at 20% power using a QSONICA Q125 sonicator and centrifuged at 14,000 × g. A BCA assay kit (Thermo Fisher Scientific) was used to determine protein concentration according to the manufacturers instructions. 50 μg of protein was loaded for each sample, and PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used to approximate molecular weight. Following transfer to nitrocellulose, the membrane was blocked in 5% milk in TBS-T (15mM Tris pH 7.4, 150mM NaCl, 0.1% Tween 20) and probed with rabbit anti-p22phox (1:50) and mouse anti-α-tubulin DM1a (1:2000) in TBS-T overnight. Infrared-labeled secondary antibodies, goat anti-mouse 800 (Rockland Immunochemicals Inc.) and goat anti-rabbit 680 (Invitrogen) (1:10,000) were used for detection on a Li-Cor Odyssey infrared scanner (Li-Cor Biosciences, Lincold, NE).