FIGURE 2.

ZAP-70 is essential for the recruitment of WAVE2 to the TCR site. A, ZAP-70-deficient Jurkat cells (P116) 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, P116 cells reconstituted with wild-type ZAP-70 expressing YFP-WAVE2 were treated as described above. C, colocalization analysis between WAVE2 and Tyr(P) was performed in P116 cells in comparison with P116 reconstituted with ZAP-70 WT by calculating Pearson's colocalization coefficients. Imaging analysis was performed on 25 cells for each experimental group. D, Jurkat E6.1 T cells expressing YFP-WAVE2 were stimulated for 2 min (+) or left unstimulated (−). After stimulation, cells were lysed and immunoprecipitated (IP) with anti-GFP. Immunoprecipitates were separated by SDS-PAGE, transferred, and blotted (IB) with anti-pZAP-70 and GFP antibodies. E, lysates of unstimulated (−) and stimulated (+) PBLs were immunoprecipitated with anti-WAVE2 and blotted for pZAP-70 and WAVE2. F, LAT-deficient T cells (J.CaM2.5) and J.CaM2.5 cells reconstituted with LAT WT, SLP-76-deficient T cells (J14), and J14 cells reconstituted with SLP-76 WT were stimulated, lysed, and immunoprecipitated with anti-WAVE2 and blotted for pZAP-70 and WAVE2. Data shown are representative of at least three independent experiments. Error bars, S.E.