ZO-1 regulates the endothelial actomyosin distribution. (A–C) Cells transfected with nontargeting (Control) siRNA or siRNAs directed against ZO-1 were analyzed by immunoblotting for ZO-1 and α-tubulin expression (A), or processed for immunofluorescence microscopy using antibodies against ZO-1 (B) or β-actin, myosin IIA, or double-phosphorylated MLC2 (C). (D) Cells transfected with siRNAs were analyzed by immunoblotting for single- and double-phosphorylated, as well as total, MLC2, and, as a loading control, α-tubulin. (E) Cells that had been transfected with siRNAs as indicated were additionally transfected with GFP or GFP-mZO1, a fusion protein constructed with a mouse ZO-1 cDNA, 24 h before analysis. The cells were then fixed and stained as indicated to monitor loss of stress fibers and increased junctional staining of F-actin and myosin upon ZO-1 reexpression. Bars: (A–C) 40 µm; (E) 20 µm.