Recombinant His-VRK1, GST-AMPKα2, GST–AMPKα2-KD (K45R), His-VRK-1, GST–AAK-2 isoform b, and GST–AAK-2 isoform b-KD (K116R) proteins were expressed in E. coli BL21 (DE3). GST-AMPKα2 and GST–AAK-2 were purified using glutathione Sepharose 4B (GE Healthcare Life Sciences, MA, USA). His-VRK1 and His-VRK-1 were purified with Ni–nitrilotriacetic acid agarose (Invitrogen, CA, USA) following the manufacturers’ instructions. For nonradioactive in vitro kinase assays using human VRK1 and AMPK, purified His-VRK1 was incubated with recombinant GST-AMPKα2 in the kinase assay buffer [60 mM HEPES (pH 7.4), 10 mM MgCl2, and 10 mM MnCl2] and 200 μM ATP for 30 min at 30°C. The enzyme reaction was stopped by the addition of 5× SDS loading buffer by boiling at 95°C for 5 min. Samples were immunoblotted with antibodies against GST (91G1, Cell Signaling Technology, MA, USA), His (2365S, Cell Signaling Technology, MA, USA), or p-AMPKα (Thr172) (2531S, Cell Signaling Technology, MA, USA). The nonradioactive in vitro kinase assays for C. elegans VRK-1 and AAK-2 was performed similarly to those for the human VRK1 and AMPK. For radioactive in vitro kinase assay, purified His-VRK1 was incubated with recombinant GST-AMPKα2 in kinase assay buffer [60 mM HEPES (pH 7.4), 10 mM MgCl2, and 10 mM MnCl2] and [γ-32P]ATP for 30 min at 30°C. The reaction was stopped by the addition of 5× SDS loading buffer by boiling at 95°C for 5 min. Radioactive signal was detected by using an x-ray film (Agfa, Antwerp, Belgium).

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