Figure 3

AIP positively regulates CBM complex formation and NF-κB activity in Jurkat T cells. (A) AIP knockdown does not affect CARMA1 Ser645 phosphorylation. Jurkat T cells were transfected with GFP or AIP targeting siRNAs 1 and 3 and stimulated with CD3/CD28 as indicated. After anti-CARMA1 IP, CARMA1 phosphorylation was detected by a phospho-Ser645-specific antibody. (B, C) AIP is required for CARMA1-BCL10 association. Jurkat T cells were transfected with siRNAs targeting GFP or AIP (siAIP1, 2, 3) and stimulated with P/I (B) or siAIP1 and 3 and stimulated with CD3/CD28 (C) as indicated. Cells were lysed and subjected to anti-BCL10 IP. (D) AIP knockdown impairs IKK T-loop phosphorylation. Jurkat T cells were transfected with siRNAs as in (B) and after P/I stimulation subjected to anti-IKKα IP. After Western Blotting, T-loop phosphorylation was detected with a phospho-specific antibody. (E) IκBα phosphorylation and degradation is reduced after AIP knockdown. Jurkat T cells were transfected with siRNAs as in (A) and stimulated by CD3/CD28 co-ligation as indicated. IκBα phosphorylation and degradation was analyzed by western blotting. (F) AIP knockdown diminishes NF-κB activation. Jurkat T cells were transfected with siRNA against GFP or siAIP1 and 2 and stimulated with anti-CD3/CD28 antibodies as indicated. NF-κB DNA binding was assessed by EMSA. (G) AIP does not influence TNFα induced NF-κB activity. Jurkat T cells were transfected with siRNAs against GFP and AIP (siAIP1), respectively, and stimulated with TNFα or CD3/CD28 for the indicated time points. NF-κB DNA binding was assessed by EMSA.