Fig. 13.

Effects of overexpression of the phosphorylation-deficient mutant of moesin (Thr558Ala) on thrombin-induced cytoskeletal remodeling. A: EC were transfected with empty vector (control), V5 (a peptide GKPIPNPLLGLDST, recognized by an antibody)-tagged wild-type (WT), or mutant (Mut) moesin, which were then detected via immunoblotting with V5 Ab. Results of scanning densitometry of Western blots are shown as % of moesin relative to control. Immunoblotting with β-actin Ab was used as a normalization control. B: after transfection with vectors expressing moesin (wild-type or mutant) tagged with V5, EC were grown on glass coverslips as described in materials and methods followed by thrombin treatment (0.5 U/ml, 5 min). EC were subjected to double immunofluorescent staining with Texas red phalloidin to visualize F-actin (B and C, top) and V5 Ab (B and C, bottom). Overexpression with mutant moesin abolishes thrombin-induced F-actin stress fibers and induces cortical actin formation (C, image g) compared with EC overexpressed with wild-type moesin (C, image c). Arrow indicates cell transfected with mutant moesin EC (C, image g). Images are representative of 3 independent experiments. Scale bar = 10 μm.