FIGURE 3.

Identification of RE1. A, schematic of the constructs assayed. B and C, Jurkat T cells in which CARD11 was stably knocked down 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. B, two-tailed unpaired Student's t test with unequal variance resulted in p values <0.008 for the values obtained under unstimulated conditions with the following constructs as compared with that observed with CARD11 ΔΙD: Δ483–671, Δ530–671, Δ568–671, Δ611–671, and Δ649–671. C, same test resulted in a p value <0.02 for the values obtained under unstimulated conditions with Δ494–671 as compared with that observed with CARD11 ΔΙD, and p values <0.006 for the comparison between Δ494–671 and either Δ494–671 M1 or Δ494–671 M2. D and E, HEK293T cells were transfected with the same amounts of each expression vector used in B and C, and lysates were probed by Western blot 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.