Fig. 3. FAK^Tyr861 associates with vß5 integrin upon Mer activation. (A) Cells were transfected with plasmid DNA encoding FAK and ß5 integrin as well as empty vector, CDMer or CDMer/KD as indicated. Subsequently, cell lysates were subjected to immunoprecipitation with anti-vß5 mAb (P1F6), and immunoblotting with anti-FAK Ab to detect FAK/vß5 integrin complex. The blot was stripped and reprobed with anti-phospho-FAK at Tyr⁸⁶¹. (B) FAK^Tyr861 binding to vß5 integrin is linked to the p130^CAS/CrkII/Dock180 complex. ß5CS-1 cells were transfected with empty vector, CDMer or CDMer/KD. CS-1 cells without transfection serve as control (-). The cell lysates were immunoprecipitated with anti-p130^CAS and analyzed by immunoblotting with anti-phosphotyrosine mAb. The blot was stripped and reprobed with anti-phospho-FAK at Tyr⁸⁶¹ to detect active FAK-p130^CAS complex. (C) Gas6 induces a complex between FAK^Tyr861 and vß5 integrin. Empty vector (lane 1), wild-type Mer (lanes 2 and 3), or Mer/KD (lane 4) was expressed with FAK in control CS-1 (lane 1) or ß5CS-1 cells (lanes 2-4) for 48 hours. After starvation for 18 hours, the cells were stimulated with 150 nM Gas6 for 10 minutes (lanes 3 and 4). The cell lysates were analyzed by immunoblotting with antibodies specific for Tyr397/FAK and Tyr861/FAK (i). The lysates were also immunoprecipitated with anti-vß5 (P1F6) mAb, and immunoblotted with anti-FAK mAb or anti-phospho-FAK at Tyr⁸⁶¹ to detect the phosphorylated and total FAK recovered with vß5 integrin immunoprecipitates (ii).