Fig. 1

UBXN7 interacts with RNF111 and RNF165 Arkadia-like RING domains. a Schematic representation of RNF111 showing its characterized domains: the three SUMO-interacting motifs (SIM) and C-terminal RING domain. The sequence of the RING core is indicated below with the cysteines and histidines of the RING-H2 domain in bold. Mutation of the first cysteine of the RING domain is annotated in red. A schematic representation of the “cross-brace” zinc finger structure of the RING domain is shown [12]. b Workflow for the identification of RING specific RNF111 partners. HEK-293 cells were transfected with GFP, GFP-RNF111-WT, or GFP-RNF111-CA before GFP-Trap affinity purification and proteomics mass spectrometry analysis. The number of peptides identified for UBXN7 in each condition is shown below. c Endogenous UBXN7 interacts with RNF111-WT but not with RNF111-CA. Whole-cell lysates (Input) and GFP-Trapped lysates (GFP-Trap) from HEK-293 cells transfected with GFP, GFP-RNF111-WT, or GFP-RNF111-CA were analyzed by western blotting with anti-GFP and anti-UBXN7 antibodies. The arrows indicate the protein of interest. d Schematic representation of Flag-tagged constructs expressing RNF111 (human) or RNF165 (rat) used in this study. The sequence alignment of the RING core is indicated below. Identical amino acids are highlighted in yellow. The RING core is 80% identical between RNF111 and RNF165. Note that the RING core sequence of RNF165 is identical in rat and human. The sequence alignment with human PIRH2, another RING-H2, is also indicated. e, f UBXN7 specifically interacts with RNF111 and RNF165 RING domains. Lysates from HEK-293 cells transfected with HA-UBXN7-WT and the indicated Flag-tagged RNF111, RNF165, or PIRH2 constructs were immunoprecipitated with Flag antibody and analyzed by western blotting with anti-Flag and anti-HA antibodies. The corresponding whole-cell lysates (Input) were analyzed with anti-HA antibody. The arrows indicate unmodified RNF111 and RNF165 proteins