Two distinct pools of the ADAP/SKAP55 module were identified in T cells. (A) Human primary T cells were left untreated or stimulated with CCL21 for the indicated time points. Immunoprecipitations were performed using anti–SKAP55 or (B) anti–ADAP antibodies, and precipitates were analyzed by Western blotting using the indicated antibodies. One representative experiment of 2 is shown. (C-D) Purified T cells were stimulated as described in panel A, and lysates were subjected to anti-RAPL (C) or anti-RIAM (D) immunoprecipitation. Precipitates were analyzed by Western blotting using the indicated antibodies. One representative experiment of 3 is shown. (E-F) Primary human T cells were subjected to 4 sequential immunoprecipitations with anti–RAPL (E) or anti–RIAM (F) antibodies, respectively, followed by final immunoprecipitations using either anti-RIAM or anti-RAPL antibodies. Precipitates were analyzed byWestern blotting with the indicated antibodies. One representative experiment of 2 is shown.