FIGURE 1.

CARD11 ID contains more than one redundant repressive element. A, schematic of the constructs assayed. B, Jurkat T cells in which CARD11 was stably knocked down (KD-CARD11) or control cells expressing a control shRNA (NT) were transfected with CSK-LacZ and Igκ₂-IFN-LUC in the presence of expression vectors for the indicated Myc-tagged CARD11 variants and stimulated with anti-CD3/anti-CD28 cross-linking for 5 h as indicated. A two-tailed unpaired Student's t test with unequal variance resulted in p values >0.05 for the values obtained under unstimulated conditions with the following constructs as compared with that observed with wild-type CARD11: Δ441–488, Δ483–535, Δ530–573, Δ568–616, and Δ649–671. C, HEK293T cells were transfected with the same amounts of each expression vector used in B, and lysates were probed by Western blot (WB) using anti-Myc primary antibody to indicate the relative expression level of each variant. β-Galactosidase activity, driven by CSK-LacZ, was used to calculate equivalent amounts of lysate for Western analysis. D, KD-CARD11 Jurkat T cell transfections in B were scaled up 9-fold, and each Myc-tagged CARD11 variant was immunoprecipitated (IP) by anti-Myc antibody and assayed by Western blot using anti-CARD11 antibody as described under “Experimental Procedures.” β-Galactosidase activity, driven by CSK-LacZ, was used to calculate equivalent amounts of lysate for Western analysis.