FIG. 7.
Regulation of binding between CARMA1 and Bcl10 via Ser109 phosphorylation. (A and B) 293T cells were transfected with 2 μg of Flag-Bcl10 construct and 0.5 μg of Myc-CARMA1 or its S109A mutant (A) or its S109D mutant (B). The total amount of DNA was equalized with empty vectors. The cells were lysed 18 h later. Monoclonal anti-Flag antibody was used for immunoprecipitation (IP). Monoclonal anti-Myc antibody or anti-Flag antibody was used for blotting. Results are representative of three independent experiments. (C and D) Jurkat cells (C) or CARMA1 mutant Jurkat cells (D) were electroporated with NF-κB reporter, pRL-TK construct, and Myc-CARMA1 or its S109A mutant. Eighteen hours later, the cells were incubated with anti-CD3 antibody (1 μg/ml) for a further 6 h, and NF-κB luciferase activities were determined. Four independent experiments were performed. Bars represent standard errors. (E) 293T cells were transfected with 1 μg of Flag-Bcl10, 0.5 μg of Myc-CARMA1, and 2 μg of CaMKII1-290. The total amount of DNA was equalized with empty vectors. The cells were lysed 18 h later. Monoclonal anti-Flag antibody was used for IP. Monoclonal anti-Myc antibody or anti-Flag antibody was used for blotting. (F) Jurkat cells were electroporated with 10 μg of siRNA D1488 or G371 constructs or vector and 5 μg of Flag-CARMA1 construct. Three days later, the cells were incubated with anti-CD3 antibody (5 μg/ml) for 10 min, lysed, and immunoprecipitated with anti-Flag antibody. Total lysates were immunoblotted with anti-Flag or anti-Bcl10 antibody. Results are representative of three independent experiments. WT, wild type; Unstim., unstimulated; Stim., stimulated.