PMC:3062687 / 30248-30982 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3062687","sourcedb":"PMC","sourceid":"3062687","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3062687","text":"In Vitro Kinase Assay\nKinase-active recombinant IKKβ and IKKα proteins were purchased from Active Motif and Millipore, respectively. Bacterially purified glutathione S-transferase (GST), GST-IκBα (1-54), wild type, mutant GST-RPS3, or RPS3 proteins were used as substrates. The in vitro kinase assay was performed as previously described29. Briefly, enzyme (100 ng) and substrate (2 μg) were co-incubated in IKK reaction buffer (25 mM Tris–HCl [pH 8.0], 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM Na3VO4, 1 mM ATP) or NleH1 reaction buffer (50 mM Tris-HCl [pH 7.6], 5 mM MgCl2, 1 mM DTT, 1 mM ATP) with 0.5 μCi 32P-γ-ATP (GE Healthcare) added at 37 °C for 30 min. 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