DP regulates spatial organization of EB1. (A and B) Immunolocalization of endogenous DP and EB1 imaged by confocal microscopy (A) and SIM (B). (C) PLA of DP with EB1 in SCC9s (top) and in SCC9s rinsed with saponin detergent to extract cytoplasmic proteins (bottom), with corresponding immunofluorescence. The graph shows quantification of DP-EB1 PLA, or for PLA of each antigen with nonspecific IgG controls (IgG control images not depicted). (D) Single time frames from time-lapse recordings of EB3-mRFP expressed in cells stably expressing DP-GFP. White lines show representative traces of EB3 paths on MTs (see Videos 1 and 2). (E) Immunolocalization of EB1, DP, Pg, and nuclei (DAPI) in Ct or DP KD SCC9s. Radar chart: EB1 comet angles categorized for frequencies between 0 and 90° relative to cell–cell contacts (broken lines, identified by Pg staining; n > 2,400 comets from three independent experiments). (F) Membrane fluorescence intensities of DP during recruitment to cell–cell contacts (calcium switch) and at steady-state in Ct or EB1 KD cells (see Fig. S2, A and B; n = 20 regions of cell–cell contact for each condition, from one of two independent experiments). All data are from SCC9s. Bars, 10 µm (except for the SIM image, in which they are 5 µm). Error bars indicate SEM.