Figure 4.

SHP2 activity increases focal adhesion kinase (FAK) phosphorylation on select residues. (A–C) LMVECs were treated with LPS (1 μg/ml, 2 h) or thrombin (1 U/ml, 15 min). (D–F) LMVECs were pretreated with SHP2 inhibitor, NSC-87877 (100 μM), or vehicle (H₂O) for 3 hours, followed by treatment with S1P (1 μM, 30 min) or methanol vehicle. FAK phosphorylation status was assayed by subjecting the lysates to immunoblot analysis, probing with antibodies directed against phosphorylated Y³⁹⁷ (A and D), Y⁵⁷⁶ (B and E), and Y⁹²⁵ (C and F) residues. Membranes were then stripped and reprobed for total FAK protein. Data are presented as a ratio of phosphorylated FAK to total FAK, mean (± SD). (A–C) n = 4; (D–F) n = 5. Insets are representative immunoblots for each experiment. *P < 0.05 treatment versus respective vehicle; ^†P < 0.05 S1P versus vehicle.