UBXN7 over-expression causes HIF1α accumulation in a UIM-dependent manner. (A) Non-ubiquitylated HIF1α accumulates in cells over-expressing wild-type Flag-UBXN7, but not in cells expressing a UIM-deleted version. 10 μM MG132 was added two hours prior to cell lysis to facilitate HIF1α detection. UBXN7 over-expression had no effect on CRL2 subunit levels. (B) Over-expression of another UIM-containing protein, PSMD4, does not alter HIF1α levels. (C, D) UIM-dependent accumulation of non-ubiquitylated HIF1α upon UBXN7 over-expression is also observed in the absence of proteasome inhibition (right panels). Wild-type UBXN7 interacted with HIF1α at various degrees of ubiquitylation, while UIM-defective UBXN7 (upon UIM deletion or point mutation at Ser297) only interacted with slow-migrating, poly-ubiquitylated HIF1α (left panels). The corresponding Flag, CUL2 and p97 western blots for the experiment in (C) are shown in figure 3D. (D) UBXN7 mutated at Ser288 within the UIM motif binds CUL2 (left panel) and causes HIF1α accumulation (right panel), similar to the wild-type protein. The indicated proteins were detected using specific antibodies.