Anti rabbit irdye 800

Western immunoblots were obtained from total protein extracts as described previously [21, 22] . Briefly, treated cells in 6-cm dishes were washed with ice-cold phosphate-buffered saline (PBS) containing 2 mmol/l sodium orthovanadate. Cells were collected by scrapping in 1 ml cold PBS, centrifuged at 13,000 rpm for 5 min at 4 C and the supernatants were discarded. Cells pellets were re-suspended in 50 μl lysis buffer, vortexed, and incubated at 4 C for 30 min followed by centrifugation at 13,000 rpm for 15 min at 4 C. The supernatants were collected, and the concentrations of the supernatant were measured with a BCA protein assay kit (Pierce, Rockford, lL, USA). Proteins were resolved on a 12% SDS-PAGE by loading 25 μg protein and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, (Bedford, MA, USA). The membranes were incubated with blocking buffer (1% casein in PBS) (Bio-Rad, Hercules, CA, USA) for 60 min at room temperature (RT). The membranes were incubated with primary antibody overnight at 4 C. Proteins were visualized using an infrared-emitting conjugated secondary antibodies anti-mouse 680 Alexa Fluor (Molecular Probes, Eugene, OR, USA) or anti-rabbit IRDYE 800 (Rockland Immunochemicals, Gilbertsville, PA, USA) diluted 1:15,000 for 60 min. Band intensities were quantified using Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA).