Integrin α3 activation initiates LL5 accumulation and LL5–integrin α3 association. (A–D) An activating antibody against integrin α3 (clone P1B5) induces cell aggregation (A; Symington et al., 1993) and LL5 accumulation at the cell cortex. (B) Suspended cells (1 × 10⁶) were preincubated with 5 µg of anti–integrin α3 antibody P1B5 in 1 ml MEGM, cultured on coverslips for 3 h, fixed, and stained with the indicated antibodies. (C) The relative fluorescence intensities along the white line in B were plotted. The arrows and asterisk indicate the plasma membranes and cell attachment sites, respectively. (D) Z-stack images of cells were collected with an LSM510 confocal microscope. All the fluorescent signals show significant accumulation at the artificial cell attachment sites. (E) Laminin-5–conjugated beads induce the accumulation of LL5s. Cells were stained with the indicated antibodies. DIC, differential interference contrast. (F) Extracts from HEK293T cells ectopically expressing RFP-LL5β and GFP-tagged cytoplasmic tails of integrins α3 (variant a) and α6 (variant a and b) were subjected to immunoprecipitation (IP) with an anti-GFP antibody and analyzed by Western blotting (WB). Bars: (B and D) 5 µm; (E) 10 µm.