Original ContributionKeap1 modification and nuclear accumulation in response to S-nitrosocysteine
Section snippets
Materials
Fetal bovine serum was obtained from HyClone (Logan, UT, USA) and minimal essential medium was from Sigma (St. Louis, MO, USA). All other cell culture reagents were from Gibco BRL (Grand Island, NY, USA). Tissue culture plasticware was obtained from Nunclon (Fisher Scientific, Raleigh, NC, USA). Spermine NONOate was from Cayman (Ann Arbor, MI, USA). Tris-glycine SDS–PAGE gels, Lipofectamine reagent, N′-(3-maleimidylpropionyl)biocytin (MPB), and NeutrAvidin–HRP were from Invitrogen (Carlsbad,
Keap1 modification by spermine NONOate and CSNO
These experiments investigated Keap1 modification by NO-related species using an assay developed by our laboratory to measure modification of specific proteins [34]. After exposure to the agents, cells were lysed in the presence of NEM to block reduced thiols. After removal of unreacted NEM, lysates were treated with DTT to reduce disulfide bridges, S-nitrosothiols, sulfenic acid derivatives, and other reversible thiol modifications. Subsequently the unreacted DTT was removed and lysates were
Keap1 modification
Results from this study clearly demonstrate that Keap1 thiols were modified in response to NO and CSNO. This is the first report of Keap1 thiol modification by reactive nitrogen species in an intact cell model. Similar responses were observed in HEK293H cells overexpressing Keap1 and in HEK293H cells containing only endogenous Keap1. It should be noted that the response to NONOate was much less pronounced than the response to CSNO. There are several possible explanations for this observation.
Acknowledgments
This work was supported by NHLBI HL61377(A.R.W.) and NHLBI HL42444 (A.R.W.).
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