Novel Binding Partners and Differentially Regulated Phosphorylation Sites Clarify Eps8 as a Multi-Functional Adaptor
Figure 6
Eps8 and IRS4 interact in an FGF2 dependent manner that correlates with an increase in their tyrosine phosphorylation.
A) HEK293T cells transfected with Myc-Eps8 were stimulated with 20 ng/ml FGF2 for 15 min either following 30 min pretreatment with SU5402 or dasatinib or in the absence of inhibitors. Anti-Myc immunoprecipitation and whole cell lysate samples were analysed by western blotting. B) HEK293T cells were stimulated with 20 ng/ml FGF2 for different lengths of time and tyrosine phosphorylated proteins were immunoprecipitated. Anti-pY immunoprecipitation and whole cell lysate samples were analysed by western blotting. C) HEK 293T cells were stimulated with 20 ng/ml FGF2 for 15 min and immnoprecipitations carried out using antibodies to either Eps8 or rabbit IgG. Resulting IP samples were analysed by western blotting. d) Following 30 min treatment with SU5402 or dasatinib and stimulation with 20 ng/ml FGF2 for a further 15 min, endogenous IRS4 was immunoprecipitated from HEK293T cells. Resulting IP samples were analysed by western blotting.
