Dok-1 is a substrate of Tec and Lyn in 293 cells.

(A) The 293 cells were transfected with expression vectors encoding Tec, Lyn, or Dok-1, or with empty vector (−). Tec immunoprecipitates were analyzed by Western blotting with antiphosphotyrosine (top panel) and anti-Tec (lower panel). Dok-1 coprecipitates with Tec, especially in the presence of Lyn. (B) The 293 cells were cotransfected with HA-tagged Dok-1 and Tec, together with (+) or without (−) Lyn. Lysates were incubated with anti-HA, anti-Dok-1, or anti-Tec, after which the precipitates were analyzed with anti-HA. Lyn stabilizes the interaction between Tec and HA-Dok-1. (C) F36P cells were stably transfected with HA-Dok-1. Tec was precipitated from lysates of nonstimulated (−) or SCF-treated (+) cells, after which the precipitates were analyzed with antiphosphotyrosine (upper panel), anti-HA (middle panel), and anti-Tec (lower panel). More HA-tagged Dok-1 is precipitated after SCF stimulation, indicating that the association of Tec and Dok-1 is stabilized within the cKit-signaling complex. (D) COS cells (upper panel) and 293 cells (lower panel) were transfected with empty vector (−) or a Dok-1 expression plasmid (+), or cotransfected with Tec and/or Lyn. Dok-1 immunoprecipitates were analyzed by Western blotting with antiphosphotyrosine (top panel). In COS cells, both Tec and Lyn can phosphorylate Dok-1, whereas in 293 cells Dok-1 is a better substrate for Tec than for Lyn.