Fig. 7

TOPORS is another E3 regulated by the UAS domain of UBXN7. a GFP-UBXN7-UAS versus GFP Interactome. Cell lysate from HEK-293 cells transfected with GFP or GFP-UBXN7-UAS were subjected to GFP-Trap affinity purification and co-immunoprecipitated proteins were subsequently identified using quantitative label-free mass spectrometry. Volcano plot illustrating the distribution of the proteins significantly enriched (fold change ≥ 2, p value ≤ 0.05, n = 5) in GFP-UBXN7-UAS compared to GFP (-Log10 (adj p value), y-axis; log₂ fold change GFP-UBXN7-UAS/GFP, x-axis). b, c TOPORS binds to UBXN7-UAS. HEK-293 cellular extracts (b) or HEK-293 cellular extracts from cells transfected with Flag-TOPORS-Nter-WT or Flag-TOPORS-Nter-C103A (c) were pulled down with the indicated GST-UBXN7 constructs and analyzed by western blotting along with the corresponding cellular extracts (input). The amount of GST proteins in each condition was revealed by stain-free as a control. d The UAS domain competes with UBE2D2 for binding to TOPORS RING domain. GST pull-down (GST-PD) of the indicated GST-RING domains with UBE2D2, in the presence or not of UBXN7-UAS. The proteins were revealed on acrylamide gel by Coomassie staining. e The UAS domain inhibits free ubiquitin chain formation induced by TOPORS RING domain in vitro. Recombinant TOPORS RING domain was incubated at 37 °C for 1 h in the presence of UBE1 (E1), UBE2D2 (E2), ubiquitin (UB), and 1 × , 5 × , or 10 × molar excess of recombinant UBXN7-UAS (UAS). Free ubiquitin chain formation was revealed by western blotting using an anti-ubiquitin (anti-UB) antibody