FIG 1.

Identification of NS5A-associated kinases. (A) AlphaScreen-based protein-protein interaction assay. FLAG-tagged full-length NS5A, and each of 404 biotinylated human protein kinases, which were synthesized in a cell-free protein production system, were mixed with the detection mixture containing anti-FLAG antibody, protein A-conjugated acceptor beads, and streptavidin-coated donor beads in 384-well plates. Luminescence signals from the mixture were detected. Ninety-five protein kinases were identified with luminescence signals of ≥1,300, of which 84 were serine/threonine kinases. The assay was performed in duplicate for each sample, and data shown are mean values of duplicate experiments. (B) Exemplary autoradiographic images of NS5A phosphorylated in vitro. Purified kinases were mixed with biotinylated NS5A proteins coupled on streptavidin beads in kinase buffer containing [γ-³²P]ATP. After the reaction, samples were subjected to SDS-PAGE and autoradiography. The arrows, black arrowheads, and white arrowheads indicate phosphorylated full-length NS5A, phosphorylated NS5A domain III, and autophosphorylated kinases, respectively.