Fig 2.

ORF36 phosphorylates ORF59 between aa 265 and 396. (A) Coomassie-stained gel of GST-ORF59 and segments used in the kinase assay. Lanes 1 and 3, ORF59-GST; lanes 4 to 6, ORF59-GST segments from aa 1 to 132, aa 133 to 264, and aa 265 to 396, respectively; lane 2, GST protein used in the assay. (B) Autoradiography image of the in vitro kinase assay performed on ORF59 and its GST-fused segments using immunoprecipitated ORF36-Flag as the kinase and the Flag vector as a control for kinase. Lane 1, GST-ORF59 as a negative control showing no phosphorylation when ORF36 was not present; lane 3, ORF59-GST phosphorylation when ORF36 kinase was present (asterisk). Among the GST-ORF59 domains, the region from aa 265 to 396 (lane 6) was phosphorylated by ORF36 (red asterisk). Lanes 2 to 6, ORF36 autophosphorylation, which is essential for its kinase activity.