FIG.1.
Time course of TNF-α-induced p47phox phosphorylation, p47phox-TRAF4 binding, p47phox-p22phox binding, and NADPH-dependent SOD-inhibitable O2− production by HMEC-1. (A) Representative immunoblots. p47phox was immunoprecipitated (IP), and then parallel membranes were immunoblotted (IB) either with a phosphoserine-specific antibody (top panel), an anti-TRAF4 antibody (second panel), an anti-p22phox antibody (third panel), or an anti-p47phox antibody (fourth panel). (B) The first three panels show quantitative densitometric analyses of immunoblots. Results are expressed as mean arbitrary absorbance units ± standard deviations obtained from three separate experiments. *, P < 0.01 compared to unstimulated cells (i.e., time zero). The bottom panel shows acute TNF-α-induced NADPH-dependent, SOD-inhibitable chemiluminescence (CL) in an HMEC-1 cell homogenate. (C) Control experiment to confirm specific capture of TRAF4 after immunoprecipitation for p47phox. CMEC from p47phox−/− mice (KO cells) were transfected with an empty vector or with p47phox cDNA. Immunoprecipitation was performed for p47phox followed by immunoblotting for TRAF4.