Anti mouse ir 680 dye

For Western blot analysis, tissues/cells were homogenized in RIPA buffer (Sigma) containing complete protease inhibitor cocktail (Roche, Basel, Switzerland) using 1.2-mm silica beads (Fisher, Hampton, NH) and the Fisherbrand Bead Mill 24 Homogenizer. Total protein homogenates were cleared by centrifugation at 12,000g for 20 minutes, and an equal amount (65 μg) of the total proteins was loaded on a 4%–12% mini gel (Invitrogen). The proteins then were transferred to a polyvinylidene difluoride membrane and probed simultaneously with anti-mouse SMVT, NF-κB, phospho NF-κB p65 (ser536) (raised in rabbit), and monoclonal β-actin antibody (raised in mouse). The blots then were incubated with anti-rabbit/anti-mouse IR 800 dye and anti-mouse IR 680 dye (LI-COR, Lincoln, NE) secondary antibodies (1:25,000) for 1 hour at room temperature. Relative expression was quantified by comparing the fluorescence intensities in an Odyssey Infrared imaging system (LI-COR) using Odyssey application software (version 3.0) with respect to corresponding β-actin.