Irdye 680lt and 800cw infrared dye coupled anti rabbit or anti mouse

Protein samples for Western blot analysis were separated by 12% SDS–polyacrylamide gel electrophoresis (PAGE) gels. Following separation on the gel, proteins were transferred using electrophoresis onto a nitrocellulose membrane and blocked at room temperature shaking in 5% milk (w/v) in tris-buffered saline (TBS) containing 0.5% Tween 20 (TBS-T). Membranes were incubated overnight at 4°C with primary antibody. Horseradish peroxidase (HRP)–tagged secondary antibodies were diluted in TBS-T/5% milk for 1 hour. Antibody-reactive bands were detected with LAS-3000, Fujifilm. Fluorescent secondary antibodies were diluted 1:10,000 in TBS-T/5% milk for 1 hour. Antibody-reactive bands were detected with the Odyssey infrared imaging system (LI-COR Biosciences). IRDye 680LT and 800CW Infrared Dye coupled anti-rabbit or anti-mouse (LI-COR Biosciences).

The following were the antibodies used: anti-mouse actin (Sigma-Aldrich, A1978), anti-rabbit BAX (Cell Signaling, 3792S), anti-rabbit BAK (Cell Signaling, 2774S), anti-rabbit acetyl alpha tubulin (Cell Signaling, 5335S), anti-rabbit HDAC6 (Cell Signaling, 7888S), anti-rabbit aldolase A (Cell Signaling, 3188S), anti-rabbit GAPDH (Cell Signaling, 5174S), anti-acetyllysine agarose (ImmuneChem, ICP0388), anti-mouse HRP secondary antibody (LI-COR Biosciences, 926-80010), and anti-rabbit HRP secondary antibody (LI-COR Biosciences, 926-80011); IRDye 680LT and 800CW Infrared Dye coupled anti-rabbit or anti-mouse (LI-COR).