Figure 8.

Mutations that disrupt SLAP dimerization also inhibit 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 three independent experiments. Refer to Fig. 7A for abbreviations of SLAP mutants.