Figure 6.

Dominant interfering mutant of SLAP inhibits TCR-mediated NFAT-AP1 activity. Jurkat T cells were transfected with 2.5 μg of the NFAT-AP1 firefly luciferase construct, 0.1 μg of thymidine kinase-driven Renilla luciferase construct, along with 20 μg of vector control or different GST-SLAP constructs. Transfectants were incubated with or without plate-bound anti-CD3 antibody (OKT3.14) for 6 hr. Cells were lysed, and both firefly luciferase and Renilla luciferase activities were determined according to the manufacturer’s instructions. The NFAT-AP1-driven firefly luciferase values were normalized based on the constitutively active Renilla luciferase activity. The data are representative of at least four independent experiments. Vector, pEBG vector; SLAP/WT, wild-type SLAP; SLAP/ΔT, 64-aa deletion at SLAP C terminus; SLAP/RK-ΔT, double mutant of SLAP containing Arg-111 to Lys and 64-aa deletion at C terminus.