1. Mapping the Pirh2 and p73 interaction sites.
Mapping the Pirh2 and p73 interaction sites. A, cell extracts prepared from Saos-2 cells ectopically expressing Myc-Pirh2 and various truncated forms of Myc-Pirh2 were analyzed by Western blot. B, the region of Pirh2 involved in binding to p73 was analyzed using a Ni2+ pull-down assay. Purified His-p73α protein was incubated with cell extracts prepared from Saos-2 cells ectopically expressing Pirh2 or Pirh2 mutants and analyzed by Western blot as indicated. An asterisk (*) indicates that the Myc-Pirh2 1–60 was unable to bind to p73. C, GST-Pirh2 and various truncated Pirh2 fusion proteins were affinity purified and analyzed by SDS-PAGE and Coomassie blue staining (top). The GST-Pirh2 proteins were mixed with purified His-p73α and analyzed by Western blot with an anti-His antibody for p73 (bottom). D, schematic representation of Pirh2 and various Pirh2 truncations. On the basis of the data provided in (B) and (C), the binding site for p73 is indicated. E, cell extracts prepared from Saos-2 cells ectopically expressing Flag-p73α and various truncated forms of Flag-p73α were analyzed by Western blot. F, the region of p73 involved in binding to Pirh2 was analyzed using a GST pull-down assay. Purified GST-Pirh2 protein was incubated with cell extracts prepared from Saos-2 cells ectopically expressing Flag-p73α or Flag-p73α mutants and analyzed by Western blot as indicated. G, schematic representation of Flag-p73 and Flag-p73 truncations. The binding site for Pirh2 is indicated and is based on the data provided in F. IB, immunoblotting.
Hong Wu et al. Mol Cancer Res 2011;9:
©2011 by American Association for Cancer Research