Nedd4 binds p73 but fails to ubiquitinate it. (A) Hek293 cells were transfected with HA-TAp73α, HA-ΔNp73α, HA-p53 or an empty vector, together with Myc-Itch or Myc-Nedd4 in the presence of MG132. Cells were subjected to IP using anti-HA antibody and analyzed by western blot, using an antibody against Myc. Itch and Nedd4 co-IP with p73. (B) Hek293 cells were transfected with HA-TAp73α, HA-ΔNp73α or HA-p53, together with Myc-Itch or Myc-Nedd4. Cell extracts were analyzed by western blot, using an antibody against HA. Expression of Itch but not of Nedd4 results in TA and ΔNp73 downregulation. (C) Cells transfected with the indicated plasmids together with a plasmid expressing Ub-HA were analyzed by western blot using an antibody against HA. Higher molecular bands characteristic of ubiquitination appear when Itch but not Nedd4 is overexpressed.