Binding between the PDZ domain of ZASP6 and p53 is direct, while interaction of p53 with ZM1 requires mediation of another protein/proteins.

<p>H1299 cells lacking endogenous p53,were transfected with pcDNA3-p53, GFP-ZASP6, GFP-PDZ and FLAG-ZM1, as indicated. Transfected cell lysates were incubated with (<b>A</b>) anti-p53 monoclonal antibody or (<b>B</b>) with anti-FLAG antibody immobilized on resin. Precipitated immune complexes were separated by SDS-PAGE. Immunoblotting was performed with anti-GFP polyclonal antibody (<b>A</b>, top panel) or anti-p53 (<b>B</b>, top panel). As controls, cell lysates were probed with rabbit polyclonal anti-GFP antibody (<b>A</b>, middle panel), monoclonal antibody to FLAG (<b>B,</b> middle panel) and anti-p53 monoclonal antibody (<b>A</b> and <b>B</b>, bottom panels). <b>C)</b> GST-overlay assay with purified His-p53 (4 μg), separated by SDS PAGE, transferred to Immobilon P membrane and overlaid with GST-ZASP6, GST-PDZ, GST-ZM1 or GST alone. GST proteins bound to p53 was detected by anti-GST polyclonal antibody. In the bottom panel, an identical membrane was probed with an anti-Histidine antibody to demonstrate that the same amount of His-p53 protein was loaded.