FIGURE 5:

Cortactin is a novel binding partner of Dock4. (A) Schematic diagrams of cortactin constructs used in this study. NTA, N-terminal acidic region; Repeat, tandem repeat of 37–amino acid actin-binding domain; α-heli, α-helical region. Numbers indicate amino acid position in the sequence. (B) Cell lysates from HEK293T cells transfected with Flag–Dock4 were used in pull-down assay with GST or GST-fused cortactin proteins. (C) Cell lysates from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with anti-Myc antibody, and bound proteins and lysates were analyzed by immunoblotting. (D) Cell lysates from cultured hippocampal neurons at 16 DIV were immunoprecipitated with anti-Dock4 antibody or control immunoglobulin G, and bound proteins and total cell lysates were analyzed by immunoblotting with anti-Dock4 and anti-cortactin antibodies. (E) Cultured hippocampal neurons at 15 DIV were fixed and double stained with anti-Dock4 (green) and anti-cortactin (magenta) antibodies. Arrowheads indicate the dendritic spines where cortactin colocalized with Dock4. Scale bar, 5 μm. (F) Fluorescence intensity profiles along cyan line drawn in E were measured in corresponding channels.