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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.
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