Figure 2.

Dynamics of RhoA activity, endothelial contractile machinery, and cytosolic Ca²⁺. EC monolayers were exposed to thrombin (Thr; 0.3 IU/mL) for different time points or vehicle (0 min; control) as indicated and samples were collected in Laemmli buffer and subjected to Western blot analysis for (A) MYPT1 phosphorylation using a phosphospecific antibody to MYPT1 (Thr850) and (B) MLC phosphorylation using a phosphospecific antibody to MLC (S18/T19). GAPDH was used as loading control. Mean ± SEM of three experiments of independent cell preparations; *P < 0.05 versus C (0 min). (C) EC monolayers were exposed to thrombin (Thr; 0.3 IU/mL) in the absence or presence of Rock inhibitor Y27632 (10 μmol/L) or vehicle (control) as indicated and albumin flux (permeability) was measured. Mean ± SEM of three experiments of independent cell preparations; *P <0.05 versus control, ^#P <0.05 versus Thr alone. (D) Cytosolic Ca²⁺‐levels (Fura‐2 ratio) was measured as detailed in methods. The graph shows collective mean data of at least 100 cells from one measurement. Representative graph of three experiments from independent cell preparation. (E) MLC phosphorylation. EC monolayers were exposed to thrombin (Thr; 0.3 IU/mL) for different time points or vehicle (0 min; control) in the absence or presence of BAPTA‐AM or ML‐7 as indicated. Representative blots from three experiments with independent cell preparation. (F) EC monolayers were exposed to thrombin (Thr; 0.3 IU/mL) in the absence or presence of ML‐7 (10 μmol/L) or BAPTA‐AM (10 μmol/L) or vehicle (control) as indicated and albumin flux (permeability) was measured. Mean ± SEM of three experiments of independent cell preparations; *P <0.05 versus control, ^#P <0.05 versus Thr alone. (G) MYPT1 phosphorylation. EC monolayers were exposed to thrombin (Thr; 0.3 IU/mL) for different time points or vehicle (0 min; control) in the absence or presence of BAPTA‐AM as indicated. Representative blots from three experiments with independent cell preparation.