Comparison of LL5α and -β functions in MCF-10A cells. (A) Domain compositions of LL5α and -β. FHA, forkhead-associated domain; cc, coiled-coil domain; PH, PH domain. (B) Myc3-tagged constructs used for rescue experiments. Bars indicate silent mutations conferring siRNA resistance. For live cell imaging, fluorescent protein-tagged versions were used. (C) Extracts from HEK293T cells ectopically expressing myc3-tagged LL5s and GFP-tagged CLASPs were subjected to immunoprecipitation (IP) with an anti-myc antibody and analyzed by Western blotting (WB). (D) At 3 h after seeding on coverslips, MCF-10Aeco cells were fixed and stained with the indicated antibodies. LL5s exhibit similar distributions at the migrating edges of cells and colocalize with CLASPs (a and b). Microtubule plus ends visualized with EB1 (c) are concentrated in the region with CLASP/LL5 accumulation. The boxed areas in c are magnified in d. (E) The colocalized fluorophores in D (a–c) were quantified. The percentages of colocalized pixels and the Pearson’s correlation coefficients in colocalized volume (R(r)) between the two color channels inside the whole cell area and the LL5-concentrated area are presented. (F) The mean fluorescence intensities of tubulin, LL5s, and EB1 staining in D (c) were compared between the LL5-concentrated area and other area. Bars: (D, a–c) 10 µm; (D, d) 5 µm.