PAK1 can phosphorylate aPKCζ in vitro. HA-aPKCζ wild-type (lanes 1 and 2), HA-aPKCζ kinase dead (lanes 3 and 4), or a GFP control (lanes 5 and 6) were transfected to HEK-293 cells and immunoprecipitated. The immunoprecipitate was subjected to an in vitro kinase assay in the presence (lanes 1, 3, and 5) or absence (lanes 2, 4, and 6) of recombinant GST-PAK1. The proteins were then separated on SDS-PAGE, stained with Coomassie brilliant blue (bottom panel), and exposed to autoradiography (top panel). The results are representative of three separate experiments.