*4.4. In Vitro Phosphorylation of Recombinant FRS2*

In vitro phosphorylation experiments were performed with recombinant proteins. One microgram of a fusion protein was incubated with kinases and 40 μCi/mL [γ-33P] ATP in reaction buffer (25 mM HEPES, pH 7.5, 20 mM MgCl2, 1 mM Na2MO3, 20 mM sodium β-glycerophosphate, 1 mM DTT, 5 mM EGTA) at 30 ◦C for 30 min. The proteins were separated by SDS-PAGE, electroblotted, and analyzed by autoradiography. Equal loading was ensured by membrane staining with Coomassie Blue.
