Fig. 5

Binding of the UAS domain impedes RING-E2 interaction. a The UAS domain competes with RING-E2 but not with RING-UB interaction. GST pull-down (GST-PD) of GST, GST-UB (upper panel) or GST-UBE2D2 (lower panel) with recombinant RNF111 RING, in the presence or not of 10 × molar excess of recombinant UBXN7-UAS. The proteins were revealed on acrylamide gel by Coomassie staining. The unbound panel shows the unbound proteins in the flow-through. Recombinant UB (in the upper panel) and UBE2D2 (in the lower panel) were used as a positive control for binding competition. b UBE2D2 binding to GST-RNF111-RING in the presence of increasing amount of UBXN7-UAS. GST pull-down (GST-PD) of GST-RNF111-RING-WT with equimolar concentration of UBE2D2 in the presence of 1 to 5 × molar excess of UBXN7-UAS. The UBE2D2 input and the unbound proteins in the flow-through are shown. c The UAS domain inhibits the binding of RNF111-RING with different known interacting E2s. GST pull-down of different GST-E2s (UBE2D1-3, UBE2E1-3, UBE2K, UBE2N, and UBE2W2) with RNF111-RING-WT in the presence or not of 2 × molar excess of UBXN7-UAS. The unbound proteins in the flow-through are shown