For western blot analysis, CD4+ T cells were stimulated and total cell lysates were obtained in lysis buffer containing 0.15M NaCl, 10mM HEPES, 0.1mM EDTA, 0.1mM EGTA, 1mM NaF, 1mM Na3VO4, 10mM KCl, 0.5% NP-40, and protease inhibitor cocktail (10%, vol/vol) (Sigma-Aldrich, St. Louis, MO).
Proteins (20 µg/lane) were then boiled at 95°C in the presence of LDS sample buffer and 2-mercaptoethanol (Life Technologies, Carlsbad, CA), subjected to SDS PAGE and then transferred to Immun-blot PVDF membranes (Bio-Rad, Hercules, CA). Membranes were blocked for 30 minutes in 3% BSA and 0.05% Tween 20 in PBS and incubated overnight with the appropriate primary antibodies, then washed and incubated for 1 hour at room temperature with the correspondent anti-mouse or anti-rabbit IgG-HRP secondary antibody (Jackson Immunoresearch, West Grove, PA). The activity of membrane-bound peroxidase was detected using the ECL system (Thermo Scientific, Waltham, MA).
Proteins (20 µg/lane) were then boiled at 95°C in the presence of LDS sample buffer and 2-mercaptoethanol (Life Technologies, Carlsbad, CA), subjected to SDS PAGE and then transferred to Immun-blot PVDF membranes (Bio-Rad, Hercules, CA). Membranes were blocked for 30 minutes in 3% BSA and 0.05% Tween 20 in PBS and incubated overnight with the appropriate primary antibodies, then washed and incubated for 1 hour at room temperature with the correspondent anti-mouse or anti-rabbit IgG-HRP secondary antibody (Jackson Immunoresearch, West Grove, PA). The activity of membrane-bound peroxidase was detected using the ECL system (Thermo Scientific, Waltham, MA).