Anti phospo p65 nf κb antibody

DCs were stimulated with 10 µg/ml CpG1826, R848 or both for 24 h and proteins were extracted in fresh lysis buffer (pH 7.9) containing 10 mM KCl, 2 mM MgCl2, 0.5 mM dithiothreitol (DTT), 1% IGEPAL CA-630, 0.2 mM ethylenediaminetetraacetic acid (EDTA) in 10 mM Hepes, 2.5 mM sodium fluoride, 0.5 mM sodium orthovanadate and protease inhibitor cocktail (Sigma-Aldrich, Cat#P8340). Following centrifugation at 12000 g for 30 min, the supernatant was recovered. Protein concentration of each sample was determined by the Bradford protein assay (Bio-Rad). 10-20 µg of protein were size-fractionated in 12% sodium dodecylsulphate-polyacrylamide gel (PVDF) (BIO-RAD, Cat#162-0177), then electrotransferred to polyvinyl difluoride membranes. Blots were blocked for 2 h in 5% bovine serum albumin (BSA)-Trisbuffered saline-0.1% Tween 20 at room temperature and incubated overnight with anti-phospo-p65-NF-κB antibody (Santa Cruz, Cat# 33039) and β-actin (Sigma-Aldrich, Cat# A2066). This was followed by incubation with anti-rabbit secondary antibody-HRP (Millipore, Cat# AP103P) for 1 h. Immunoreactivity was detected by enhanced chemiluminescence (ECL plus, Amersham Biosciences). Blots were scanned with an Image Quant 300 imager and analyzed by Image Quant TL software (Amersham Biosciences, GE Healthcare). Phospho-p65-NF-κB intensity was normalized with the corresponding β-actin blot.

DCs were incubated with CpG1826 or R848 for 24 h, then fixed with 2% PFA washed with PBS and incubated overnight with anti-phospo-p65-NF-κB antibody (Santa Cruz, Cat# 33039). Then, DCs were incubated with anti-rabbit secondary antibody-FITC (Vector Cat# FI-1000) for 1 h. Immunofluorescence signal intensity per cell was measured using ImageJ software.