FIGURE 3.

LAT is required for the recruitment of WAVE2 to the TCR site. A, LAT-deficient T cells (J.CaM2.5) expressing YFP-WAVE2 (green) were plated onto stimulatory antibody-coated coverslips and then fixed and stained for phosphotyrosine (pTy-Red). Far left, differential interference contrast (DIC); far right, fluorescence intensity scatter plots. B, J.CaM2.5 cells reconstituted with LAT WT and expressing YFP-WAVE2 were treated as described above. C, colocalization analysis between WAVE2 and Tyr(P) performed in J.CaM2.5 cells in comparison with J.CaM2.5 reconstituted with LAT WT by calculating Pearson's colocalization coefficients (scatter diagrams). D, FRET efficiency between LAT and WAVE2 was determined in J.CaM2.5 cells expressing LAT-CFP and YFP-WAVE2 throughout TCR activation. Imaging analysis was performed on 30 cells for each experimental group. E, PBLs were co-stimulated for 2 min with anti-CD3 and -CD28 antibodies, lysed, and immunoprecipitated (IP) with anti-WAVE2 and blotted (IB) for pLAT and WAVE2. Data shown are representative of at least four independent experiments. Error bars, S.E.