GKAP interacts with DIC and is required for DIC interaction with Dlg1 and DIC recruitment to microtubules. (A) Astrocyte extracts were submitted to immunoprecipitation with anti-DIC (IP DIC) or irrelevant control (IP Ctl) antibodies. Immunoprecipitates and total cell lysates were analyzed by Western blotting (WB) using anti-GKAP and anti-DIC antibodies. (B) Extracts from primary astrocytes were obtained at the indicated times after wounding. Lysates were subjected to immunoprecipitation with anti-Dlg1 (IP Dlg1) or irrelevant control antibody (IP Ctl). Immunoprecipitates and total cell lysates were analyzed by Western blotting (WB) using anti-DIC (top panels) and anti-Dlg1 antibodies (bottom panels). (C) Anti-GKAP, anti-tubulin, and anti-DIC Western blots of protein extracts from astrocytes 5 d after nucleofection by a control siRNA (si Scramble) and three different GKAP siRNAs (si GKAP 1, si GKAP 2, si GKAP 3). (D) Western blots showing DIC and Dlg1 in Dlg1 immunoprecipitates from astrocytes nucleofected with control (si GFP, si Scramble) or GKAP (si GKAP 1) siRNAs. (E) TIRF images showing DIC (top) or DIC (red) and microtubules (green) (middle) in GKAP-depleted cells. Colocalization is shown in yellow (bottom). (F) Quantification of colocalization of DIC on microtubules in astrocytes nucleofected with si GKAP 1 and si GKAP 2. Dashed lines indicate the orientation of the wound. Bars, 5 µm.