FIG. 1.

CARMA1(L808P) is functionally defective. (A) JPM50.6 cells were transfected with plasmids encoding an NF-κB-dependent luciferase gene in the presence of expression plasmids encoding the wild-type CARMA1 [CARMA1(WT)] or L808P mutant [CARMA1(L808P)]. Twenty-four hours after transfection, the transfected cells were stimulated with (i) media, (ii) 1 μg of anti-CD3 and 1 μg of anti-CD28 per ml, or (iii) 10 ng of PMA and 1 μg of anti-CD28 antibodies per ml for 6 h. The cells were lysed, and luciferase activity was determined. All of the samples were also transfected with an EF1α promoter-dependent Renilla luciferase. The constitutively expressed Renilla luciferase activity was used to normalize the transfection efficiency. All data are presented as fold induction. The expression levels of the transfected CARMA1 were detected by Western blotting with anti-Myc antibodies (inset). Unstim, unstimulated. (B) JPM50.6 cells stably expressing Myc-tagged wild-type CARMA1 or CARMA1(L808P) were unstimulated (a and c) or stimulated with PMA (10 ng/ml) plus anti-CD28 antibodies (1 μg/ml) (b and d) for 6 h. The cells were collected and examined for the expression of NF-κB-dependent GFP in these cells by fluorescence-activated cell sorting (FACS). The y axis indicates the cell numbers (10⁴), and the x axis indicates the fluorescence intensity.