Mutation of 14-3-3ζ binding sites of SLP76 and GADS leads to additive increase in SLP76 microclusters and NFAT activation. (A) J14 cells were transiently transfected with SLP76-YFP, -WT, or -S376A together with CFP-GADS constructs, either WT or a T254A mutant. Cells were stimulated for 5 min on anti-CD3–coated coverslips, fixed, and imaged by confocal microscopy. The number of SLP76 microclusters was automatically assessed in selected cells showing homogeneous levels of YFP and CFP fluorescence. A similar analysis was performed on phospho-LAT microclusters (Fig. S5 A). Horizontal lines and error bars show means and SEM. (B) J14 cells stably expressing FLAG-SLP76 (J14-WT) or FLAG-SLP76-S376A (J14-S376A) were transiently cotransfected with the indicated CFP-GADS construct and an NFAT-luciferase reporter plasmid. Cells were stimulated with the indicated concentrations of plate-bound anti-CD3 and 1 µg/ml of soluble anti-CD28 mAbs for 5 h. Control samples were stimulated with PMA and Ca²⁺ ionophore to assess maximal NFAT-dependent transcription. Luciferase activity was measured in cell lysates. Data represent means ± SD of normalized luciferase activity from quadruplicate samples expressed as a percentage of maximal luciferase activity. Comparable expression levels of all constructs are shown in Fig. S5 B.