Properties of AMAP1 and PRKD2 binding. (A–G) GST-tagged AMAP1 fragments (A and E) or AMAP1 PRD fragments bearing the indicated mutations (G) were each incubated in vitro with lysates of Cos7 cells expressing full-length Xpress-PRKD2. GST-tagged PRKD2 fragments (C) were each incubated in vitro with lysates of Cos7 cells expressing full-length EGFP-AMAP1. Coprecipitation of GST fusion proteins with PRKD2 or AMAP1 was analyzed by immunoblotting using anti-Xpress (B, F, and G) or anti-EGFP (D) antibodies, respectively. In B, D, F, and G, amounts of GST fusion proteins used were analyzed by Ponceau S staining. WCL, whole cell lysate (5 µg); GST, GST alone used as a control. (H–J) The GST-C1 fragment was overexpressed in MDA-MB-231 (H), Hs578T (I), and MDA-MB-435s cells (J), and their adhesion to collagen (Adhesion) and Matrigel chemoinvasion (Invasion) was measured. Cells overexpressing GST alone or the GST-C4 fragment were included as controls. Data are presented as percentages calculated by normalizing the values obtained for the GST-expressing control cells as 100%. 1,617 ± 212 (∼5.39%), 836 ± 96 (∼3.34%), and 457 ± 58 (∼2.17%) MDA-MB-231, Hs578T, and MDA-MB-435s cells expressing GST (and mVenus as a marker), respectively, were calculated to have transmigrated per 6.4-mm-diam Matrigel-coated Boyden chamber filter under these conditions. Data are shown as mean ± SEM of triplicate experiments (error bars).