Figure 3.

Analysis of contractile signaling activated by thrombin. (A) Thrombin-induced Rho activation was assessed by RhoGTP pulldown assay. (B) Cytoskeletal remodeling in response to thrombin treatment (0.2 U/ml, 10 min) was analyzed by immunofluorescence staining for F-actin (red) and myosin A (green). Merged images depict areas of actin and myosin colocalization, appearing in yellow. (C) Immunofluorescence analysis of F-actin (red) and vinculin (green) localization in control and thrombin-treated ECs. Areas of colocalization appear in yellow. (D) HPAECs were pretreated with vehicle, Y-27632 (2 µM, 30 min), or Janus kinase (JAK) inhibitor I (5 µM, 30 min), followed by stimulation with thrombin. Myosin light chain (MLC) phosphatase (MYPT) and MLC phosphorylation was analyzed by Western blotting with the corresponding antibody. Probing for β-actin was used as a normalization control. (E and F) HPAECs were pretreated with Y-27632 (E) or blebbistatin (15 µM, 30 min) (F) followed by stimulation with thrombin, and TER was measured over time. (G) Quantitative analysis of paracellular gap formation in control and thrombin-treated HPAECs with or without Y-27632 or blebbistatin (Bleb) preconditioning. Data are expressed as mean ± SD; n = 3; *P < 0.05 versus thrombin. (H) HPAECs were pretreated with JAK inhibitor I (5 µM, 30 min) followed by stimulation with thrombin, and TER was measured over time. Arrows in panels E, F, and H indicate time point of thrombin. Jak-I, Janus kinase inhibitor; Yi, Y-27632.