Specificity of phosphospecific antibody, and phosphorylation of C3G on Y504 upon coexpression with Src family kinases. Cos-1 cells were transfected with the expression constructs for Hck (A) or c-Src (B) along with C3G as indicated and western blotting of whole cell lysates was performed using the phosphospecific antibody pY504. The blots were reprobed with C3G, Hck and anti pTyr (Panel A) or Src (Panel B) to show their expression in the lysates. pY504 C3G and pTyr was detected by ECL and C3G, Hck and Src by alkaline phosphatase dependent color development. UT indicates untransfected cell lysates. Y504F is a mutant of C3G in which tyrosine 504 is replaced by phenylalanine.