Dendritic Cell Protein Analysis

DC were washed with calcium- and magnesium-free PBS and lysed in 1 mL of lysis buffer containing 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P40, 50 mM NaF, 1 mM sodium orthovanadate, dithiothreitol, and a cocktail of protease inhibitors (cOmplete EDTA-free, Roche). Whole cell lysates were prepared and submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 5%  β-mercaptoethanol using 12% polyacrylamide gels. Harvest of DC and preparation of whole cell lysates were performed as previously described [27 (link)]. Proteins were transferred to polyvinylidene difluoride membranes and immunolabeled using primary antibodies against IκBα, phospho-IκBα, and β-actin. The bound primary antibodies were detected using secondary anti-mouse or anti-rabbit antibodies (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and ECL chemiluminescence detection kit (PerkinElmer, Waltham, MA, USA). The protein bands were scanned and digitized, and the density of each band was determined using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).