Fig 3.

EGLN3 selectively inhibits IKKγ ubiquitination. (A) HeLa cells were treated with 10 ng/ml of TNF-α for the indicated times prior to harvest. Cell lysates were immunoprecipitated with anti-IKKγ. The immunoprecipitates and cell extracts were analyzed by immunoblotting with the indicated antibodies. (B) HeLa cells were transfected with control siRNA (−) or EGLN3 siRNA (+). At 60 h after transfection, cell lysates were prepared after treatment of cells with 10 ng/ml of TNF-α for 10 min and analyzed as described for panel A. (C) HeLa cells were transfected with HA-IKKγ, myc-Ub, and EGLN3 siRNA. Cell lysates were analyzed as described for panel A. (D) HEK293T cells were transfected with a plasmid expressing EGLN3 or an empty vector. At 30 h after transfection, cells were then treated with 10 ng/ml of TNF-α for different times. Cell lysates were analyzed as described for panel A. (E) HEK293T cells were transfected with HA-IKKγ, myc-Ub, and different amounts of FLAG-EGLN3. Cell lysates were immunoprecipitated with anti-HA and then immunoblotted with anti-myc. Cell lysates were examined by immunoblotting. (F to I) HEK293T cells were transfected with the indicated plasmids. Cell lysates were immunoprecipitated with anti-HA (F, G, and H) or anti-FLAG (I). The immune complex was then immunoblotted with anti-myc (F and H) or anti-Ub (G and I). To monitor expression of the transfected genes, cell lysates were examined by immunoblotting with the indicated antibodies. IP, immunoprecipitation; IB, immunoblotting; Ig H, heavy chain of IgG.