FIG. 12.
Hypoxia-promoted Sumo-2/3 conjugation to IκBα prevents its degradation. (A) U2OS cells were treated with the proteasome inhibitor MG132 3 h prior to a 30-min treatment with 1% O2 or 10 ng/ml TNF-α. Cells were harvested directly into SDS loading buffer, and IκBα levels were determined by Western blotting (WB). (B) U2OS cells were treated with TNF-α for the indicated periods in the presence or absence of 1% O2 prior to lysis. Whole-cell lysates were analyzed by Western blotting. (C) HEK293 cells were transfected with 1 μg of IκBα and 1 μg of His6-ubiquitin, and where indicated, they were treated with TNF-α or MG132, or both. Ubiquitinated proteins were purified using Ni2+ agarose, and pulldown eluates were analyzed by Western blotting for IκBα. Arrows indicate modified versions of IκBα. (D) HEK293 cells were transfected with 1 μg of IκBα and 1 μg of a His-tagged version of ubiquitin or Nedd8 where indicated. Cells were treated or not with 1% O2 for 15 min and were harvested 48 h posttransfection. Extracts were processed and analyzed as for panel C. Arrows indicate modified versions of IκBα. (E) HEK293 cells were transfected with 1 μg of IκBα and 1 μg of a His-tagged version of Sumo-2 (S2) or Sumo-3 (S3) where indicated. Cells were treated or not with 1% O2 for 15 min and were harvested 48 h posttransfection. Extracts were processed and analyzed as for panel C. Arrows indicate modified versions of IκBα. (F) Sumo-2 is conjugated to lysine 21 of IκBα. HEK293 cells were treated with1 μg of wild-type (Wt) or K21A or Y42A mutant IκBα and were exposed or not to 1% O2 for 15 min prior to lysis. Sumoylated proteins were purified as for panel C, and pulldown eluates were analyzed for IκBα by Western blotting. Arrows indicate modified versions of IκBα.