GKAP binds Dlg1 and is recruited to leading edge microtubules after wounding. (A) Astrocyte extracts were immunoprecipitated with anti-GKAP (IP GKAP) or irrelevant control (IP Ctl) antibodies. Immunoprecipitates and total cell lysates were analyzed by Western blotting (WB) using anti-GKAP and anti-Dlg1 antibodies. (B) Immunofluorescence of GKAP (red) and microtubules (green) in confluent, nonmigrating astrocytes. (C) GKAP immunostaining in astrocytes 0 and 6 h after wounding. GKAP is recruited to the leading edge of migrating cells (arrowheads). Dotted lines show cell edges. (D) Quantification of GKAP recruitment to the cell leading edge 0, 1, 4, and 6 h after wounding. Results are shown as mean ± SEM of 300 cells from three independent experiments. (E) TIRF images of GKAP (red) and microtubule (green). Colocalization is shown in yellow (right panels). Arrows in B and C point to the centrosome region. Dashed lines indicate the orientation of the wound. Bars: (B and C) 10 µm; (E) 5 µm.