Fig. 4.

N4BP1 directly inhibits protein ubiquitylation through competition with substrates for Itch binding. (A) In vitro self-ubiquitylation activity of ΔC2Itch was tested in the absence (lane 2) or in the presence (lanes 5–7) of increasing doses of bacterially purified GST-N4BP1. The catalytic-defective ΔC2Itch-C830A mutant (lane 1) and free GST (lane 4) were used as negative controls. Reaction mixtures were analyzed by IB with anti-Ub and anti-GST antibodies. (B) (Upper) Competition experiments were conducted using GST-ItchΔC2 fusion protein bound to glutathione-Sepharose resin and incubated with a constant amount of in vitro-translated HA-p73α and N4BP1-V5 at 6-fold excess. The beads were washed and subjected to SDS/PAGE and IB with anti-p73 and anti-N4BP1 antibodies. (Lower) Binding of p73 to Itch was normalized to the amount of glutathione-Sepharose resin as assessed by anti-GST IB. (C) GST fusion proteins of the individual WW domains of Itch were bound to glutathione-Sepharose beads and incubated with in vitro-translated HA-p73α. Protein complexes were analyzed by IB analysis using anti-p73 antibody.