Anti irf4 antibody

Briefly, total Tfh cell lysates were prepared by lysing cells in Triton lysis buffer supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (10 mM NaF and 1 mM Na₃VO₄). IRF4 protein in the cell lysates was immunoprecipitated using anti-IRF4 antibody (Santa Cruz Biotechnology, 5 μg per sample) followed by detection of Batf by immunoblotting with anti-Batf antibody (Santa Cruz Biotechnology, 1:500 dilution).

BMDMs were lysed with 1× sample buffer (Tris [pH 8], 2% SDS, 10 mM EDTA, 0.01% bromophenol blue, 25 mM 1,4-dithio-DL-threitol, and 250 mM 2-ME), heated to 95°C for 10 min and separated by 10% sodium dodecyl sulphate–PAGE. Proteins were then transferred to 0.45-μm PVDF membranes (Millipore, Billerica, Massachusetts, USA), and membranes were washed and blocked with 5% skim milk in PBS. Antibodies were incubated overnight at 4°C in 5% BSA 0.05% Tween-20, and 0.1% sodium azide PBS. Membranes were washed three times (10 min) before addition of secondary HRP-coupled antibody in 5% skim milk PBS for 1 h at room temperature. PICOlucent (Gbioscience, St Louis, MO, USA) was then added, and the film (Agfa, Septestraat, Mortsel, Belgium) was exposed and developed. Anti-phospho IκBα, anti-IκBα, anti-phospho p65, anti-p65, anti-IRF5 and anti-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) and were used at 1/2000 dilutions. Anti-IRF4 antibody was obtained from Santa Cruz Biotechnology. The secondary polyclonal anti-mouse or -rabbit IgG HRP-coupled antibody was used at a 1/5000 dilution (Molecular probe, Eugene, Oregon, USA). Bands were quantitated and normalized with actin using ImageJ software.