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. 2015 Jun;52(6):695–707. doi: 10.1165/rcmb.2013-0489OC

Figure 6.

Figure 6.

FAK regulates endothelial barrier function through SHP2-mediated mechanism. (A, B, D, and E) Equal numbers of LMVECs were transfected with eukaryotic vectors encoding catalytically active SHP2 (SHP2D61A), GFP–FAK-related nonkinase (FRNK), or GFP cDNAs. (A) At 48 hours, LMVECs were treated with FAK inhibitor, PF-573228 (0.1 μM), or vehicle (H2O) for 3 h. FAK phosphorylation status was assayed by subjecting the lysates to immunoblot analysis, probing with antibodies directed against phosphorylated Y397 residue. Membranes were then stripped and reprobed for total FAK protein. Representative blot is shown (n = 4). (B) Representative plot of changes in monolayer resistance were measured in LMVECs overexpressing FRNK (square symbols) or GFP (diamond symbols) cDNA, in the presence (open symbols) and absence (closed symbols) of PF-573228 (0.1 μM). Arrow indicates point of addition (n = 3). FRNK protein overexpression was confirmed by immunoblot analysis of the lysates of transiently transfected LMVECs with an antibody raised against FAK (inset). Representative images are shown (n = 3). (C) LMVECs were treated with FAK inhibitor, PF-573228, or vehicle (H2O) for 3 h. Equivalent amounts of lysate were immunoprecipitated for SHP2 using an antibody derived from rabbits. Immunocomplexes were resolved with total lysate via SDS-PAGE and immunoblotted with a FAK antibody derived from mice. Membranes were then stripped and reprobed with an SHP2 antibody, and FAK–SHP2 association was determined by the ratio of FAK bound to total immunoprecipitated SHP2. Inset: representative immunoblots. Data are presented as means (± SD); n = 3. *P < 0.05 versus vehicle. (D) At 48 hours, LMVECs were treated with FAK inhibitor, PF-573228, or vehicle (H2O) for 3 hours. SHP2 was immunoprecipitated from lysates and subjected to FAK immunoblot analysis as for (C). Inset: representative immunoblots. Data are presented as means (± SD). *P < 0.05 versus vehicle. (E) Changes in monolayer resistance were measured in LMVECs overexpressing SHP2D61A (squares; n = 4) or GFP (diamonds; n = 5) cDNA, in the presence (open symbols) and absence (closed symbols) of PF-573228 (0.1 μM). Arrow indicates point of addition. Data are presented as means (± SD). *P < 0.05 versus GFP vehicle.