The association of DOCK2 and CrkL in vivo.

(A) Analysis of the association of CrkL with DOCK2 in 293T cells. The pCAGGS-CrkL and pCXN2-Flag-DOCK2 were transiently transfected, and an immunoprecipitation assay was performed by anti-CrkL Ab (lane 2) or normal rabbit serum (NRS; lane 1). Immunoblotting was performed with the use of anti-Flag Ab (top panel) and anti-CrkL Ab (bottom panel). Total cell lysates appear in lane 3. (B) Analysis of the association of c–Crk-II with DOCK2 in 293T cells. The sets of cotransfected plasmid were as follows: pCAGGS–c–Crk-II and pCXN2 vector (lanes 1, 3, and 4); pCAGGS–c–Crk-II and pCXN2-Flag-DOCK2 (lanes 2, 5, and 6). Cell lysates were analyzed by immunoprecipitation with the use of anti-DOCK2 (αDK2) Ab or NRS (lanes 3-6). Total cell lysates are shown in lanes 1 and 2. (C) Association of CrkL and DOCK2 in Jurkat cells with stimulation. Cells were in suspension (sus; lanes 1 and 2), plated on collagen type I–coated dishes (collagen; lanes 3 and 4), or plated on poly-L-lysine–coated dish (poly-L-K; lanes 5 and 6). Cells were incubated for 15 minutes at 37°C, and cell lysates were immunoprecipitated with anti-DOCK2 (αDK2) Ab (lanes 2, 4, and 6) or NRS (lanes 1, 3, and 5), and precipitates were analyzed by immunoblotting with antibodies, indicated below each panel. Total cell lysates appear in lane 7. The arrowheads indicate DOCK2 in the top panel, Vav in the second panel, CrkL in the third panel, and c–Crk-II in the bottom panel. (D) Association of endogenous CrkL and DOCK2 in Jurkat, MOLT4, and Raji cells. Cell lysates were immunoprecipitated by anti-CrkL Ab (αCrkL; lane 3, 9, and 13) or anti-DOCK2 Ab (αDK2; lane 6, 10, and 14) and probed with anti-DOCK2 Ab (top panels) and with anti-CrkL Ab (bottom panels).