Cortactin directly interacts with shootin1 in axonal growth cones. (A) Coimmunoprecipitation of shootin1 and cortactin in COS7 cells. Cells were cotransfected with myc-shootin1 and FLAG-cortactin, and lysates were incubated with anti-FLAG antibody. The immunoprecipitates were immunoblotted with anti-FLAG or anti-myc antibody. (B) In vitro binding assay using purified shootin1 (2 µM) and purified FLAG-cortactin (2 µM). Proteins were incubated with anti-FLAG antibody, and the immunoprecipitates were then analyzed by SDS-PAGE and CBB staining. 0.125% of the input proteins were also analyzed. As reported previously (Wu and Parsons, 1993; MacGrath and Koleske, 2012a), purified cortactin is composed of a single major band at 80 kD and an additional upper band (blue arrowheads). (C) Coimmunoprecipitation of endogenous shootin1 and cortactin from rat (postnatal day 6) brain lysates. Brain lysates were incubated with anti-shootin1 antibody or control IgG. The immunoprecipitates were immunoblotted with anti-shootin1 or anti-cortactin antibody. (D) Rat hippocampal neurons (3 d in vitro) costained with anti-cortactin (red) and anti-shootin1 (green) antibodies. Arrows and arrowheads denote an axonal growth cone and minor process growth cones, respectively. (E) Fluorescence images of an axonal growth cone labeled with anti-cortactin (red) and anti-shootin1 (green) antibodies. Enlarged views of the filopodia in the white square are shown to the right. Arrowheads indicate cortactin colocalized with shootin1. Bars: (D) 20 µm; (E, main panels) 10 µm.