FIG. 2.

Activation of wild-type Fes induces localization to MTs in vivo. COS-7 cells were transiently transfected with expression vectors for wild-type GFP-Fes (WT), kinase-active GFP-Fes (L145P), kinase-dead GFP-Fes (K590E), or the empty vector (vector) as a control. Cells were also cotransfected with a combination of wild-type or kinase-dead GFP-Fes and an activated form of the Src family kinase, Hck-YF. Forty-eight hours after transfection, the cells were fixed and stained with α-tubulin, Hck, and/or Fes-tyrosine phosphospecific antibodies. (A) Confocal images of a representative cell coexpressing wild-type GFP-Fes and Hck-YF. Overall Fes protein distribution was visualized by GFP fluorescence. Secondary antibodies conjugated with Cy3 or Cy5 were used to differentiate tubulin from active Fes (pFes) staining. GFP, Cy5, and Cy3 fluorescences are represented as green, blue, and red, respectively, while merged colors appear white. (B) (Top) Cells from each transfection condition were examined for the appearance of MT-like localization of each GFP-Fes protein. For cotransfected cultures, only cells exhibiting GFP-Fes and Hck protein expression by immunofluorescence were counted. At least 100 cells were counted for each condition. The error bars represent standard deviations. (Bottom) Immunoblot analysis of GFP-Fes phosphotyrosine content (pTyr; top), protein level (GFP; middle), and Hck protein expression (bottom).