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. 2004 Jun 7;101(24):8945–8950. doi: 10.1073/pnas.0400588101

Fig. 4.

Fig. 4.

Cys-179 of IKKβ is target for S-nitrosylation. (A) C10 cells were transfected with wt or C179A HA-IKKβ, treated with 1 mM GSNO/l-cys or l-CSNO for 15 min, before exposure to 10 ng/ml TNFα for 5 min. IKK activity was assessed in an in vitro kinase assay, after immunoprecipitation with an HA antibody. IB, anti-HA immunoblot. (Bottom) Quantitation by phosphoimage analysis. Results are expressed as percentage of IKK activity compared with TNFα-only-treated cells. (B Left)wt(Upper) or C179A HA-IKKβ-transfected C10 cells (Lower) were treated with 1 mM GSNO/l-cys for 15 min before exposure to 10 ng/ml TNFα for 5 min. S-nitrosylated proteins were immunoprecipitated, by using an S-nitrosocysteine antibody (IP SNO) and IKKβ detected by detection of HA by Western blotting. (Lower) HA Western blots on total cell lysates. (B Right) Assessment of specificity of the S-nitrosocysteine antibody. wt (Upper) or C179A HA-IKKβ transfected cells (Lower) were left untreated (left lane), treated with 1 mM l-CSNO for 15 min (middle lane) or treated with 1 mM l-CSNO for 15 min followed by incubation with HgCl2 (right lane) before immunoprecipitation. S-nitrosylated proteins were then immunoprecipitated by using an S-nitrosocysteine antibody (IP: SNO) and IKKβ detected by Western blotting for HA; (Lower) HA Western blots on total cell lysates.