v-crk specifically interacts with and enhances the tyrosine phosphorylation status of the 3' portion of the Cas SD chimera in cells (SrcKM/Cas(SD)).a) Cos-7 cells were transfected with vector coding for the SrcKM/Cas(SD) chimera alone (lane 1), or along with mutants of v-crk, WT (lane 2), SH2 mutant (lane 3) or SH3 mutant (lane 4). The SrcKM/Cas(SD) chimera was then immunoprecipitated with antibodies to HA and probed with anti-phosphotyrosine (top panel). The blot was then stripped and then probed with antibodies to HA to detect the SrcKM/Cas(SD) chimera (middle panel). The bottom panel shows the expression of the v-crk mutants in the transfection as determined by anti-gag blotting. Arrows point to the SrcKM/Cas(SD) chimera and v-crk in the top, middle and bottom panels respectively. b) Cos-7 cells were transfected with the following plasmids, and immunoprecipitated with anti-HA antibodies. SrcKM/Cas(5'SD) chimera (lane 1), SrcKM/Cas(3'SD) chimera (lane 2), and SrcKM/Cas(SD) chimera (lane 3) alone, or along with WT v-crk (lanes 4–6). The top panel was probed with antibodies against phosphotyrosine, and the bottom panel with antibodies against HA to detect the chimeras. Probing whole cell lysates with antibodies to v-crk (anti-gag) demonstrated that cells leading to lanes 4–6 expressed equivalent levels of v-crk (data not shown).