Fig 7.

EGLN3 competes with cIAP1 for IKKγ binding. (A and B) HEK293T cells were transfected with various amounts of EGLN3. Cell lysates were analyzed by immunoblotting. (C and D) HeLa cells were transfected with EGLN3 or control siRNA for 48 h. Prior to harvest, cells were treated with TNF-α (10 ng/ml) for 10 min. Cell lysates were analyzed by immunoblotting. (E, F, G, and J) The cell lysates from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with anti-FLAG. The immunoprecipitates and cell lysates were immunoblotted with the indicated antibodies. (H and I) HeLa cells were transfected with siRNA. At 48 h posttransfection, cells were treated with TNF-α (10 ng/ml) for 5 to 10 min. Cell lysates were immunoprecipitated with anti-IKKγ or control IgG. The immunoprecipitates and cell lysates were analyzed by immunoblotting with the indicated antibodies. (K) HeLa cells were transfected with EGLN3 siRNA or control siRNA. At 48 h posttransfection, cells were treated with FLAG–TNF-α for different times. The anti-FLAG immunoprecipitates and lysates were evaluated by immunoblotting. (L) HEK293T cells were transfected with the indicated plasmids. The anti-FLAG immune complex and lysates were analyzed by immunoblotting. (M) HeLa cells were transfected with EGLN3 siRNA or control siRNA. At 48 h posttransfection, cell lysates were prepared and subjected to immunoprecipitation with anti-IKKγ. The immunoprecipitates and lysates were examined by immunoblotting. IP, immunoprecipitation; IB, immunoblotting; IgH, heavy chain of immunoglobulin; IgL, light chain of immunoglobulin; *, nonspecific band.