Fig. 7.

Dose-response of Src inhibitor AZD0530 on FAK activation loop Tyr⁵⁷⁶ and Tyr⁵⁷⁶ isolated from oncogenic Src-expressing MEFs. Oncogenic Y⁵²⁹FSrc-expressing MEFs were cultured in the presence of varying concentrations of AZD0530 (0.01, 0.1, 1, 10, and 50 μM) for 12 h. FAK was isolated from cellular lysate by immunoprecipitation. LC-MRM/MS was then used to absolutely quantify the amount of FAK activation loop modification. A, the amount of modified Tyr⁵⁷⁶/Tyr⁵⁷⁷ FAK activation loop (⋄). B, the amount of FAK activation loop in the mono-phosphorylated pTyr⁵⁷⁶ (□) and bis-phosphorylated pTyr⁵⁷⁶/pTyr⁵⁷⁷ (▵) forms. C, an IC₅₀ value of Src inhibition was calculated using the amount of pTyr⁵⁷⁶/pTyr⁵⁷⁷ (▵) determined at each dose. Data points represent the means of duplicate experiments, where error bars are ± S.D.