Fig. 4. Conserved tyrosine residues in Dok-4 are important for sustained ERK and AKT activation. (A) (a) Schematic representation of Dok-4, -5 and -6 structures. Y indicates tyrosine residues, and the tyrosine residues conserved among Dok-4-6 are indicated in bold. (b) Y187, Y220 and Y279 are conserved across a wide range of species. (B) (a) Each mutant was stably expressed in TGW cells as a V5 epitope-tagged protein. Dok-4 was immunoprecipitated with an anti-V5 antibody after GDNF stimulation, followed by immunoblotting with anti-V5 or anti-phosphotyrosine antibody. (b) TGW cells and TGW cells stably expressing each mutant were stimulated with GDNF (50 ng/ml) for the indicated times, and cell extracts subjected to western blot analysis with the indicated antibodies.