The Astrin/SKAP plus-end tracking mutant alters Clasp1 localization behavior during mitosis. (A) Localization of LAP-Clasp1 C terminus in mitosis. Arrows indicate localization to cell cortex. (B) Immunoprecipitation of the LAP-Clasp1 C terminus from mitotic cells displaying identified peptides, with data pooled from two purifications with either 100 or 300 mM KCl in the buffer. (C) IF images of mitotic cells (either normal metaphase spindles or cells with a multipolar phenotype) showing the localization of Clasp1 and EB1. Clasp1 spindle and plus-end localization is reduced in the SKAP ΔEB condition. (D, left) Cocalization of Clasp1 to microtubule plus ends in early prophase cells in which endogenous SKAP is replaced by wild-type (WT) SKAP WT or the SKAP ΔEB mutant. Clasp1 localization to plus ends is eliminated in the SKAP ΔEB mutant. (right) Zoom from boxed region on the left images. (bottom) Maximum-intensity linescans of fluorescence intensity at the microtubule plus ends marked with the indicated numbers above. Intensities are plotted as percentages of the maximum intensity quantified in this image set. (E) IF images showing anti-SKAP and anti-EB1 in control cells or cells codepleted for Clasp1 and Clasp2 (HeLa Flp-In). SKAP localization to plus ends persists in this case. Bars: 5 µm; (inset/zoom) 2 µm.