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Fig. 2. Mer-mediated tyrosine phosphorylation of p130^CAS is dependent on FAK activation. (A) HEK-293T cells were transfected with FAK-expressing plasmid plus empty vector, CDMer, or CDMer/KD as indicated. After 48 hours, cell lysates were immunoprecipitated with anti-FAK mAb, and the immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine Ab (i) or antibodies specific for Tyr 397/FAK or Tyr861/FAK (ii). FAK immunoprecipitates were subjected to an in vitro kinase assay to measure FAK activity and the autophosphorylation level of FAK was analyzed by densitometry (iii). (B) Effect of mutant FAK on p130^CAS phosphorylation. HEK-293T cells were co-transfected with vector encoding p130^CAS (lanes 1-4), CDMer (lanes 2-4), in combination with 0 (lane 1), 0 (lane 2), 1 (lane 3) and 3 (lane 4) µg of plasmid encoding FAK/KD (kinase negative mutant). p130^CAS was immunoprecipitated from extracts containing equal amounts of total proteins and subjected to immunoblotting with mAb against phosphotyrosine. (C) Tyrosine phosphorylation of FAK^Tyr861 is Gas6-inducible. Mer- or Mer/KD-expressing HEK-293T cells were prepared as in Fig. 1B, and stimulated with or without Gas6, and the cell lysates were immunoprecipitated with anti-FAK mAb, and the immunoprecipitates were analyzed as in A.

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