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For TAp73/Itch and TAp73/MDMX co-IP cells were treated for 24 h with 1 µg/ml PpIX and 30 μM MG-132 was added 3 h before cell harvest. For p73/MDM2 co-IP cells were treated with PpIX or Nutlin for 24 h (HCT p53−/− cells). Cells for both whole cell lysates and immunoprecipitates were solubilized in lysis buffer: 25 mM Tris HCl, pH 8.0, 150 mM NaCl and 1% Nonidet P-40 (0.5% for co-IP). For co-IP, 1 mg protein was immunoprecipitated with 1.5 μg of α-TAp73 rabbit polyclonal antibody (Bethyl Laboratories, TX, USA) or normal mouse IgG (Millipore, MA, USA). Immuno-complexes were absorbed onto 40 μl of Dynabeads^® Protein A (Invitrogen, Sweden) for 5 h at 4 °C. The immunoprecipitates were washed with 1 ml of lysis buffer. The antibodies used for detection were: anti-p73 monoclonal antibodies [20 (link)] (IMG 246, Imgenex, UK), anti-MDM2 (Santa Cruz, Germany), anti-Itch (Calbiochem, Sweden), anti-MDMX (Bethyl Laboratories, TX, USA).
Western Blot was performed according to the standard protocol. 100 μg of total cell lysate was subjected to electrophoresis and the following antibodies were used to detect proteins: anti-TAp73 (Bethyl Laboratories, TX, USA), anti-Bax (Santa Cruz, Germany) anti-PUMA (Cell Signaling), anti-Noxa (Calbiochem, Sweden), anti-PARP1/2 (Santa Cruz, Germany), anti-actin (Sigma-Aldrich, Germany).