Lasp-1 is colocalized with actin in cell membrane extensions of human BT-474 breast and SK-OV-3 ovarian cancer cells. (A) Confocal immunofluorescent microscopy analysis of the subcellular distribution of Lasp-1 in BT-474 cells. Cells were fixed with paraformaldehyde, permeabilized, and Lasp-1 was immunostained with the pab805 polyclonal antibody and Texas Red-conjugated donkey anti-rabbit antibody. Two optical sections parallel to the cell substratum, spaced by 1 µm, are shown, from bottom (a, c) to top (b, d) of two different optical fields. The closed arrows and arrowheads show accumulation of Lasp-1 in filopodium and apical membrane extensions, respectively. Lasp-1 is not detectable at cell-cell junctions, shown by open arrowheads. Bar, 10 µm. (B–E) Double labeling of Lasp-1 and actin with (C, E) or without (B, D) cytochalasin D treatment in BT-474 (B, C) and SK-OV-3 (D, E) cells. Lasp-1 (e, h, k, n) immunostaining is as in (A). Actin (f, i, 1, o) is stained with fluorescein-phalloidin. The false color imaging represents Lasp-1 as red and actin as green and colocalization of the two proteins as yellow (g, j, m, p). cyt D, cytochalasin D. Arrows show colocalization of Lasp-1 and actin. Bar, 10 µm.