Fig 4.

The deubiquitinating enzymes A20 and CYLD are not involved in EGLN3-mediated inhibition of IKKγ ubiquitination. (A) HEK293T cells were transfected with FLAG-CYLD, or FLAG-A20, in the presence of pSuper, pSuper-CYLD shRNA, or pSuper A20 shRNA. At 48 h posttransfection, cell lysates were analyzed by immunoblotting. (B) HEK293T cells were transfected with the indicated plasmids. Cell lysates were analyzed with anti-HA, anti-FLAG, or anti-tubulin antibodies. (C) HEK293T cells were transfected with the indicated plasmids. Cell lysates were subjected to denatured immunoprecipitation with anti-HA. The immmunoprecipitates and lysates were evaluated by immunoblotting. (D and E) HEK293T cells were transfected with FLAG-tagged EGLN3 or OTUB1 or its catalytically inactive mutant C91S (OTUB1C/S). Cell extracts were prepared and labeled with ubiquitin derivatives as detailed in Materials and Methods. F, FLAG; Ub-Vs, ubiquitin vinyl sulfone; Ub-VME, ubiquitin vinyl methyl ester; Ub-Cl, chloroethyl ubiquitin; Ub-Br, bromoethyl ubiquitin; IP, immunoprecipitation; IB, immunoblotting; IgH, heavy chain of IgG; TBS, Tris-buffered saline.