Figure 4

Molecular associations in CD40 signaling complexes involved in ROS production. (A) HEK 293T cells were transfected transiently with combinations of plasmids: human CD40 plasmid with myc epitope, hCD40(myc) and either empty vector, p85(myc), or 5-LO(flag); 5-LO plasmid with either empty vector or hCD40; and p85 plasmid with either empty vector or hCD40. Analyses of the anti-CD40 immune complexes show specific associations of CD40 with p85 but not with 5-LO. In reciprocal experiments, CD40 was detected in p85 but not in 5-LO immunoprecipitates. (B) HEK 293T cells were transfected transiently with a combination of p85(myc) and either empty vector or 5-LO(flag) or vice versa. Analyses of the immune complexes of the cell lysates with anti-myc Ab show a selective association of p85 with 5-LO. Reciprocal experiments also showed associations of these molecules. The expression of the plasmids was determined by WB of the lysates using Abs against epitope tags. The data shown represent three separate experiments.