Ubiquitin- and substrate-binding to UBXN7 do not correlate with CUL2 binding. (A) Illustration of the CRL2 ubiquitin-ligase bound to its substrate HIF1α. The core subunits of CRL2 are highlighted in blue. The UBA-UBX protein UBXN7 interacts with ubiquitylated HIF1α via its UBA domain. (B) CUL2 interaction with UBXN7 is independent of ubiquitin-binding. Flag-UBXN7 was immunoprecipitated from cells thermo-sensitive for the ubiquitin-E1. When these cells were grown at the restrictive temperature for 20 hours, there was a marked reduction in protein ubiquitylation (right panel). CUL2-binding to UBXN7 was not affected by the reduced ubiquitin-binding observed under these conditions (left panel). (C) Flag-UBXN7 stably interacts with the core subunits of the CRL2 complex. Flag-UBXN7 was immunoprecipitated from HeLa cells treated or not with 10 μM MG132 for two hours. Flag-UBXN7 stably interacted with CUL2, elongin C and RBX1. The interaction with HIF1α and VHL could only be detected upon proteasome inhibition (left panel). (D) Endogenous UBXN7 stably interacts with the core subunits of the CRL2 complex. As in (C), but endogenous UBXN7 was immunoprecipitated from HeLa cells using specific antibodies crosslinked to protein A-beads. (B-D) The indicated proteins were detected using specific antibodies in the immunoprecipitates (left) and the input cell extracts (right).