FIG. 3.
(A) Dof is important to activate ERK upon FGF signaling. S2 cells transfected with myc-btlact alone or in combination with dof-V5 were lysed after induction of expression for the indicated times with 0.6 mM CuSO4. Whole-cell lysates were subjected to SDS-PAGE, and ERK phosphorylation levels were examined by immunoblot analysis with anti-dpERK antibodies. Protein levels were evaluated for myc-Btlact and Dof-V5. (B) Dof is specifically phosphorylated upon FGF signaling. S2 cells were transfected with the indicated combination of constructs and lysed after induction of expression for the indicated times. Tyrosine phosphorylation levels were investigated on whole cell extracts by antiphosphotyrosine immunoblotting (upper panel), Dof-V5 expression levels by anti-V5 immunodetection (middle panel) and activation of ERK by anti-dpERK immunoblotting (bottom panel). (C) The different Dof proteins are not phosphorylated to the same extent. S2 cells were transfected with the indicated combination of expression plasmids and lysed after 8 h of induction (+). Transfected cells left untreated for the same time were used as controls (−). Tyrosine phosphorylation of the different constructs was assayed with antiphosphotyrosine antibodies (left panel) and protein expression levels by anti-V5 immunoblotting (right panel).