Fig. 2
From: The UAS thioredoxin-like domain of UBXN7 regulates E3 ubiquitin ligase activity of RNF111/Arkadia
UBXN7 interacts directly with RNF111 RING domain through its UAS domain. a Schematic representation of the domain organization of UBXN7 and the different mutants generated, together with their binding capacity to RNF111, N8-CUL2, and VCP observed in b. b, c UBXN7 binds RNF111 via its UAS domain. Whole-cell Lysates (input) and corresponding GFP immunoprecipitated lysates (GFP-Trap) from HEK-293 cells transfected with the different GFP-UBXN7 deletion mutants were analyzed by western blotting with the indicated antibodies. The arrows indicate RNF111 and CUL2. The asterisk indicates neddylated CUL2. d The UAS domain of UBXN7 binds directly to RNF111. UBXN7-WT, UBXN7-∆UAS, and UBXN7-UAS recombinant proteins were pulled down with GST, GST-RNF111-Cter-WT, or GST-RNF111-Cter-CA. UBXN7 proteins were revealed by western blotting with anti-UBXN7 antibody. The Ponceau staining shows GST proteins. The input shows the initial amount of each recombinant UBXN7 proteins before pull-down. e The RING domain of RNF111 has a binding affinity in the low micromolar range for UBXN7-WT and its UAS domain (Kd = 2.3 and 0.9 µM, respectively) and a stoichiometry of 1:1 (N = 1). ITC experiments were performed by titration of 200 µM of RNF111 RING domain into 10 µM of UBXN7-WT or UBXN7-UAS domain, or into 15 µM of UBXN7-∆UAS at 25 °C. ND not determined. The Kd values correspond to the average of replicate experiments (UAS, n = 3; UBXN7-WT, n = 2). ITC data values obtained for each experiments are indicated in Additional file 3: Table S1. f Schematic representation of the domain organization of the UAS containing UBA-UBX proteins UBXN7, FAF1, and FAF2, together with the matrix of percent identity of their UAS domain obtained with Clustal Omega multiple sequence alignments. g The RNF111 RING domain binds specifically to the UAS domain of UBXN7. HEK-293 lysates transfected with constructs expressing HA-tagged UAS domains from UBXN7, FAF1, or FAF2 were pulled down with GST-RNF111-Cter-WT or GST-RNF111-Cter-CA and subsequently analyzed by western blotting along with the corresponding whole-cell lysates (input). GST proteins were detected with stain-free as a control