Fig. 4

UBXN7 inhibits RNF111 auto-ubiquitylation in a UAS-dependent manner. a UBXN7 inhibits RNF111 auto-ubiquitylation in cells. Lysates (input) from U2OS UBXN7-KO clone #1 transiently transfected with Flag-RNF111-WT or Flag-RNF111-CA together with HA-tagged empty vector ( −), UBXN7-WT (WT), UBXN7-∆UAS (∆UAS), or UBXN7-UAS (UAS) were immunoprecipitated with the UB pan selector resin and subsequently analyzed by western blotting. b UBXN7 inhibits RNF111 auto-ubiquitylation in vitro. Recombinant RNF111-Cter-CA or WT were incubated at 37 °C for 1 h in the presence of UBE1 (E1), UBE2D2 (E2), ubiquitin (UB), and recombinant UBXN7-WT (WT), UBXN7-∆UAS (∆UAS), or UBXN7-UAS (UAS). RNF111 ubiquitylation was revealed by western blotting using an antibody targeting the RNF111 C-terminal domain. c The UAS domain inhibits free ubiquitin chains formation induced by RNF111-RING domain in vitro. Recombinant RNF111-RING was incubated at 37 °C for 10 min in the presence of UBE1 (E1), UBE2D2 (E2), ubiquitin (UB) and 1 to 10 molar excess of recombinant UBXN7-UAS. Ubiquitin chains were revealed by western blotting using an anti-UB antibody