In vitro kinase assay

Purified FAK-FERM (100ng) or FAK-CD (100ng) were incubated with purified HER2 (100ng) or additional RTK for 30 min in the presence or absence of ATP (10µM) in standard tyrosine kinase buffer: 20mM HEPES (pH=7.3), 5mM MgCl₂, 5mM MnCl₂, 2mM DTT, 0.1mg/mL BSA, and 0.1mM Na₃VO₄. Proteins were resolved on 4–20% gradient gels and probed for phosphorylated FAK (Y397), FAK (Y925), HER2 (Y1248), FAK-FERM, FAK-CD, and HER2/RTK using standard western blotting techniques.