In vitro kinase assay

For RSK kinase assays, human recombinant-activated RSK1 purchased from SignalChem (Catalog # R15-10G) was used with bacterially purified recombinant GST-RIOK2 (aa 443–552) as substrate (WT and S483A), under linear assay conditions. Assays were performed for 10 min at 30°C in kinase buffer [25 mmol/L Tris-HCl (pH 7.4), 10 mmol/L MgCl₂, and 5 mmol/L β-glycerophosphate] supplemented with 5 μCi of [γ-³²P]ATP. All samples were subjected to SDS-PAGE followed by immunoblotting, and incorporation of radioactive ³²P label was determined by autoradiography using a Fuji PhosphorImager with ImageQuant software. The data presented are representative of at least three independent experiments.