Fig. 6. Chat is associated with the Cas-Crk signaling complex. The association of Chat with Cas or Crk was examined by a co-immunoprecipitation assay. 293T cells were transfected with (+) or without (-) expression plasmids for Chat, Cas or Crk. Transfected cell lysates were immunoprecipitated with anti-Cas or anti-Crk antibodies; co-immunoprecipitated Chat was detected by an anti-Chat immunoblot. The expression level of Chat, Cas and Crk was analyzed by total lysate immunoblot of each protein. The different mobilities of endogenous Crk (Crk) and Myc-tagged Crk (Myc-Crk) are shown in the right side of the panel. The amount of Chat co-immunoprecipitated with Cas was not affected by Crk expression level. In Chat and Crk co-expressing cells, the amount of Chat co-immunoprecipitated with Crk was not increased. When Cas was expressed together with Chat and Crk, a marked increase of Chat in Crk immunoprecipitate was observed.