Fig. 2.

N4BP1 selectively inhibits ubiquitylation of Itch substrates. H1299 cells were transfected with GFP-p73α (A), GFP-p73γ (B), PCDNA empty vector (C), or Flag-p53 (D), along with HA-tagged ubiquitin (HA-Ub), their specific E3, Itch (A–C), or MDM2 (D) in the absence or presence of N4BP1. Twenty-four hours later, cells were treated with 40 μM proteasome inhibitor MG-132 for 1 h before harvesting. Cell lysates were subjected to IP with anti-GFP (A and B), anti-c-Jun (C), or anti-Flag (D) antibodies under denaturing conditions. Substrate-ubiquitin immunocomplexes were analyzed by anti-HA IB analysis and subsequently probed with anti-GFP (A and B), anti-c-Jun (C), or anti-Flag antibodies (D).