As described previously, CD40 signaling complexes immunoprecipitated from A20.2J cells contained HOIP as well as TRAF2, TRAF3, IKKa/ b, and IKKc. The amounts of TRAF2, TRAF3, and cIAP1 in CD40 immunoprecipitates from HOIP-deficient cells were similar to those from parental A20.2J cells, indicating that HOIP is not required for the association of these proteins with CD40. In contrast, IKKa/b and IKKc were not detectable in CD40 immunoprecipitates recovered from HOIP-deficient cells. The amounts of IKKa/b and IKKc in CD40 immunoprecipitates from HOIP-reconstituted cells were similar to those detected in samples prepared from parental cells, demonstrating that the defects in IKK recruitment we observed were due specifically to the absence of HOIP expression. These data indicate that HOIP is required for recruitment of the NF-kBactivating complex to the CD40 signaling complex. As shown here and in our previous study, IKKc present in CD40 immunoprecipitates appears to have a higher molecular weight than that found in cell lysates. To confirm that this higher molecular weight species was indeed IKKc, we generated A20.2J cell lines containing a stably integrated retroviral vector that encoded an epitope-tagged version of mouse IKKc. Cell lysates and CD40 immunoprecipitates prepared from these cells were analyzed by Western blotting for the epitope tag. This analysis produced a pattern of bands that was essentially the same as that obtained using an antibody specific for native IKKc. Kinase Inhibitor Library Moreover, analysis of cells expressing epitope tagged-IKKc confirmed that recruitment of IKKc to CD40 requires HOIP. To determine what was responsible for the increased molecular weight of IKKc in CD40 immunoprecipitates, the protein samples were incubated with lambda phosphatase, which removes phosphates attached to tyrosine, threonine, or serine residues in proteins. This treatment reduced much of CD40-associated IKKc to an apparent molecular weight similar to that of the protein in cell lysates. However, at least one band of significantly higher molecular weight remained after phosphatase treatment, suggesting that CD40-associated IKKc is subject to at least one other modification in addition to phosphorylation. Overall, these data demonstrate that HOIP is required for the association of IKKc with CD40, and suggest that this event is coupled to posttranslational modifications of IKKc. Our results indicate that the protein HOIP is critical for CD40- induced signals that regulate B cell function.