Pirh2 is an E3 ligase for p73 in vitro.

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Pirh2 is an E3 ligase for p73 in vitro. Pirh2 is an E3 ligase for p73 in vitro. A and B, GST-Pirh2 was evaluated for its capacity to ubiquitinate purified His-p73α and His-p73β as indicated and analyzed by immunoblotting with anti-Ub to reveal ubiquitinated products (top) or with an antibody against p73 (ER-15) to reveal ubiquitinated p73 species. C, Pirh2 preferentially uses Ub with Lys residue at Lys-63, Lys-29, or Lys-11 for p73 ubiquitination in vitro. Affinity purified GST-Pirh2, His-p73α, or His-p73β were added to bacterial extracts containing recombinant E1 and E2 (UbcH5b) and wild-type Ub or ubiquitin mutants as indicated and analyzed by immunoblotting with anti-Ub to reveal ubiquitinated products (top) or anti-p73 (ER-15) to reveal ubiquitinated p73 species (bottom). D, similar to (C); samples after the ubiquitination reaction were immunoprecipitated with anti-p73 (ER-15) and analyzed by immunoblotting with anti-Ub (top) or anti-p73 (ER-15, top) as indicated. IB, immunoblotting. Hong Wu et al. Mol Cancer Res 2011;9:1780-1790 ©2011 by American Association for Cancer Research