Immunolocalization of phosphorylated RLC and myosin IIA in anti-β₂GPI antibody-exposed endothelial cells. Endothelial cells were incubated with medium alone, or with β₂GPI and either control or anti-β₂GPI antibodies, and then fixed and stained with RLC (P-18/19) or myosin IIA antibodies. Cells were also stained with phalloidin to study formation of the actin network, and with 4,6 diamidino-2-phenylindole to localize nuclei. (A) Assembly of the actin network (arrow, lower left panel) occurs exclusively in cells treated with β₂GPI and anti-β₂GPI antibodies, with colocalization of phosphorylated RLC and actin (arrow, lower right panel). (B) Incubation of cells with anti-β₂GPI antibodies causes actin assembly (arrow, lower left panel), as well as actin-myosin association, as demonstrated by colocalization of these 2 proteins (arrow, lower right panel). Confocal images were collected using a ×63 oil objective with 1.4 numerical aperture on a Zeiss LSM 501 microscope. Bar represents 20 μm.