Anti alpha tubulin

Western blotting, immunoprecipitations (IP), and cell fractionations were performed as described (Sen et al., 2013 (link), 2015 (link)). IPs from mitochondrial and cytoplasmic fractions were performed as described, except the IP was done in mitochondrial isolation buffer containing 0.5% Triton X-100 and 1 mM DTT. Western blots were exposed to the following antibodies: anti-Clu (1:15,000; Cox and Spradling, 2009 (link)), anti-alpha-Tubulin (1:5000, cat# AA4.3-S, Developmental Studies Hybridoma Bank, University of Iowa, IA, USA), anti-Myc (1:5000, cat# M4439, Sigma-Aldrich), anti-Complex V (CVA) (1:100,000, cat# Ab14748, Mitosciences), rabbit anti-GFP (1:10,000, cat# Ab290, AbCam), anti-PGK (1:25,000, cat# Ab113687, Invitrogen), mouse anti-FLAG (1:10,000, cat# F1804, Sigma), rabbit anti-Calreticulin (1:1000, cat# Ab2907, Abcam), mouse anti-Porin (1:2000, cat# Ab14734, Mitosciences).

CHO cells were separately transfected with either wild-type xtMC2R or xtMC2R mutant I158/A, F161/A (TM4), V172/A (TM5) or M166/A (EC2) and each can was co-expressed with cMRAP for 5 days in advance of cell lysis. Cells were homogenized in RIPA buffer and sonicated to collect total protein. Sample protein levels were then measured by nano-spectrophotometry and analyzed by poly-acrylamide gel electrophoresis. A 4% to 20% Mini-PROTEAN® TGX™ Precast Protein Acrylamide Gels (Biorad, Hercules, CA, USA), and then samples were transferred to a nitrocellulose membrane (Genesee Scientific; San Diego, CA, USA). Membrane was blotted with anti-V5 antibody (Rockland Immunochemicals, Limerick, PA, USA) at a final concentration of 1:500, and anti-alpha-tubulin from Developmental Studies Hybridoma Bank (University of Iowa) at a final concentration of 1:500. Membrane was scanned using a Protein Simple FluorChem system (San Jose, CA, USA) and analyzed using Fiji; an open-source platform for biological-image analysis. The Western analysis was done in triplicate.