Figure 6
Figure 6. Lyn kinase regulates FAK phosphorylation and the role of FAK in regulating endothelial integrity. (A) Lyn kinase was co-immunoprecipitated with FAK. HUVECs grown in 60-mm culture dishes were lysed, and the cell extracts were incubated with anti-FAK antibody followed by incubation of protein A/G beads. Immunoprecipitates and total cell lysates were analyzed by western blotting with anti-FAK and anti-Lyn antibodies as indicated. (B) FAK phosphorylation was inhibited by PP2. HUVECs were incubated with either PP2 (10 μM) or vehicle (DMSO) for 10 and 60 min, and cell lysates were immunoprecipitated with anti-FAK antibody. Phosphorylated FAK was detected by western blotting using 4G10, an anti-phosphotyrosine antibody. The same membrane was reprobed for total FAK with an anti-FAK antibody. (C) Effect of Lyn silencing on the phosphorylation of FAK. HUVECs were transfected with control or Lyn siRNA. Forty-eight hours after transfection, cells were harvested to determine the level of phosphorylated FAK as described in B. Similar results were obtained in 3 independent experiments. (D) Knockdown of FAK by transfection of specific siRNA in HUVECs. Cells were transiently transfected with indicated concentrations of control or FAK siRNA. Forty-eight hours after transfection, cells were harvested and lysed for western blot analysis. (E) Effect of FAK silencing on TER measurement. Confluent HUVECs seeded onto microelectrodes were transfected with control or FAK siRNA as indicated. Three hours after transfection, the changes in TER across HUVEC monolayers were continuously recorded for 30 hours. Recorded values are plotted as the mean from 4 independent experiments as means ± SD. (F) Disruption of AJ in FAK siRNA transfected confluent HUVEC monolayers. HUVECs were cotransfected with control or FAK siRNA together with siGLO control siRNA as indicated. Forty-eight hours after transfection, cells were fixed and immunostained for VE-cadherin. Knockdown of FAK resulted in formation of interendothelial gaps, as pointed by the arrows. (G) Quantification of gaps and (H) cell numbers in 4 random images (mean ± SD).

Lyn kinase regulates FAK phosphorylation and the role of FAK in regulating endothelial integrity. (A) Lyn kinase was co-immunoprecipitated with FAK. HUVECs grown in 60-mm culture dishes were lysed, and the cell extracts were incubated with anti-FAK antibody followed by incubation of protein A/G beads. Immunoprecipitates and total cell lysates were analyzed by western blotting with anti-FAK and anti-Lyn antibodies as indicated. (B) FAK phosphorylation was inhibited by PP2. HUVECs were incubated with either PP2 (10 μM) or vehicle (DMSO) for 10 and 60 min, and cell lysates were immunoprecipitated with anti-FAK antibody. Phosphorylated FAK was detected by western blotting using 4G10, an anti-phosphotyrosine antibody. The same membrane was reprobed for total FAK with an anti-FAK antibody. (C) Effect of Lyn silencing on the phosphorylation of FAK. HUVECs were transfected with control or Lyn siRNA. Forty-eight hours after transfection, cells were harvested to determine the level of phosphorylated FAK as described in B. Similar results were obtained in 3 independent experiments. (D) Knockdown of FAK by transfection of specific siRNA in HUVECs. Cells were transiently transfected with indicated concentrations of control or FAK siRNA. Forty-eight hours after transfection, cells were harvested and lysed for western blot analysis. (E) Effect of FAK silencing on TER measurement. Confluent HUVECs seeded onto microelectrodes were transfected with control or FAK siRNA as indicated. Three hours after transfection, the changes in TER across HUVEC monolayers were continuously recorded for 30 hours. Recorded values are plotted as the mean from 4 independent experiments as means ± SD. (F) Disruption of AJ in FAK siRNA transfected confluent HUVEC monolayers. HUVECs were cotransfected with control or FAK siRNA together with siGLO control siRNA as indicated. Forty-eight hours after transfection, cells were fixed and immunostained for VE-cadherin. Knockdown of FAK resulted in formation of interendothelial gaps, as pointed by the arrows. (G) Quantification of gaps and (H) cell numbers in 4 random images (mean ± SD).

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