FIG. 6.

Tio is tyrosine phosphorylated by Src family kinases in vivo. 293T cells were transfected with expression vectors (lane 1) or an expression plasmid for Flag-Tio (lane 2), Lck, Src, or myc-tagged Fyn kinase (lane 3) or for Flag-Tio plus the respective kinase (lane 4). Twenty micrograms of total cell lysates was separated by SDS–9% PAGE and analyzed for tyrosine-phosphorylated proteins (A) by Western blotting (W.B.). Expression of the transfected plasmids was controlled by detection of each individual kinase with specific antibodies (B) or, for Flag-Tio, with epitope-specific antibodies (C). Arrowheads to the right of the phosphotyrosine blots indicate a protein detected only after cotransfection of Flag-Tio with Lck or Src. Molecular mass standards are given on the right.