FIGURE 3.

p73 and PTEN form an interaction in vivo and in vitro. A, p73 and PTEN form a direct interaction in vitro. Recombinant GST-PTEN, GST-C124S (PTEN lipid phosphatase mutant), and His-p73α were expressed and purified from E. coli and used in GST-pulldown assays. Interactions were detected with anti-His, and pulldown efficiency was counter-blotted with anti-GST. B, p73 and PTEN interact in vivo. FLAG-PTEN and HA-tagged p73α or p73β were overexpressed in 293T cells (left panel), and immunoprecipitation (I.P.) with PTEN antibodies was performed. Blots were probed for HA and then stripped and re-probed with PTEN. FLAG-PTEN and Myc-tagged p73ϵ or p73γ were overexpressed (right panel) and immunoprecipitated with PTEN antibodies as described for the previous panel and then stripped and re-probed with Myc antibodies. C, schematic diagram of p73 isoforms. Specific protein domains are labeled throughout the diagram as follows: transactivation domain, DNA binding domain, oligomerization, and the C terminus. Positive results from PTEN binding to various p73 isoforms is denoted by plus symbols. D, PTEN domain mapping with p73. An in vitro GST pulldown assay was performed with GST-PTEN and GST(186–351) after addition of His-p73α. A Western blot was probed for anti-His and counter-blotted for anti-GST, and positive results of p73 interaction with PTEN are displayed as plus symbols.