Fig. 1. Dap160 interacts with aPKC. (A) Drosophila Dap160 and vertebrate intersectin protein domains. (B) Dap160 antibody detects two bands in wild-type lysate (top arrow, 160 kDa; bottom arrow, 120 kDa) that may correspond to the two predicted isoforms shown in A; and these bands are absent in lysate from dap160 mutants, illustrating the specificity of the antibody. An independently generated antibody gives the same result (see Fig. 2N). (C) Immunoprecipitation from larval lysate using an aPKC antibody and a control antibody (Bgal) and blotted with a Dap160 antibody shows aPKC co-immunoprecipitates Dap160 protein (arrowhead). (D,E) In vitro protein interaction experiments. (D) In vitro generated Dap160 protein coupled to glutathione S-transferase (GST) beads can bind in vitro produced aPKC protein (arrowhead). Beads alone do not bind aPKC; input lane shown at left. (E) In vitro generated Dap160 protein coupled to glutathione S-transferase (GST) beads can bind in vitro produced Par-6 protein (arrowhead). Beads alone do not bind Par-6; input lane shown at left. (F,G) Dap160 directly stimulates aPKC activity and this effect can be partially blocked by Par6. Top: presence (+) or absence (-) of each protein; middle: phosphorylation of the aPKC substrate peptide. Histogram shows quantification of the phosphorylation (bars) over a schematic depiction of protein levels (see Materials and methods for protein concentrations). Note that aPKC alone can have high activity immediately after its synthesis (F) or much lower activity after storage (G), but can still be stimulated by Dap160.