PDZ and ZM1 regions of ZASP6 directly bind Ankrd2.

<p><b>A)</b> Co-IP of ZASP6 and Ankrd2 using lysates of COS-7 cells transfected with the indicated combination of pCMV vectors expressing cMyc-Ankrd2 and FLAG-ZASP6. Immunoprecipitation was performed with anti-FLAG antibody. The presence of Ankrd2 in the immune complex was confirmed by probing the membrane with mouse polyclonal anti-Ankrd2 antibody (upper panel). As controls, the cell lysates were tested with polyclonal antibodies to anti-Ankrd2 (middle panel) and anti-ZASP6 (bottom panel). <b>B)</b> Co-IP of PDZ-ZASP6 and Ankrd2 using lysates of COS-7 cells transfected with cMyc-Ankrd2 and pEGFP-PDZ as indicated. The cell lysates were immunoprecipitated with an anti-cMyc monoclonal antibody and probed with a polyclonal goat anti-GFP antibody conjugated to HRP (upper panel). Comparable expression levels of Ankrd2 and PDZ-ZASP6, weredemonstrated by probing the membrane with a mouse anti-Ankrd2 polyclonal antibody (middle panel) and rabbit anti-GFP polyclonal antibody (lower panel). <b>C)</b> Co-IP of ZM1-ZASP6 and Ankrd2 using lysates of COS-7 cells transfected with cMyc-Ankrd2 and FLAG-ZM1 as indicated. Ankrd2 was immunoprecipitated with anti-FLAG EZview resin and detected with an anti-Ankrd2 mouse polyclonal antibody (upper panel). As controls, cell lysates were tested with an anti-Ankrd2 mouse polyclonal antibody (middle panel) and with an anti-FLAG rabbit polyclonal antibody (bottom panel). All secondary antibodies were conjugated with HRP. <b>D)</b> GST-overlay assay with purified recombinant proteins expressed in <i>E. coli</i>. His-Ankrd2 (4 μg) was separated by SDS-PAGE, blotted and membrane strips were incubated with 4 μg of either GST-ZASP6, GST-PDZ, GST-ZM1 or GST alone, washed and then probed with anti-GST goat polyclonal antibody. As a control, an anti-histidine antibody was used to probe membrane strips identical to those used for the overlay to show that the His-Ankrd2 protein was equally loaded (lower panel). <b>E)</b> A schematic diagram of ZASP6 showing the positions of the PDZ domain (aa 11–84) and ZM-motif (aa 148–173). In this study the expression vectors with constructs for PDZ and ZM1 (ZASP6 without the PDZ domain) expressed respectively 85 and 197 amino acid fragments of ZASP6.