FIG. 4.
Purification scheme used to identify NS1O-activating protein kinases. HeLa cell replication extracts were analyzed for protein kinases which are able to phosphorylate and activate NS1O for RCR in a kinase-free system. Chromatography steps 1 (phosphocellulose) and 2 (anion exchange) allow the bulk separation of protein kinases. Steps 3 (protamine) and 4 (hydroxylapatite) are performed to further purify members of the PKC family. Selective elution of the fractions under investigation from rows 1, 2, and 3 was achieved with the indicated NaCl concentrations, while the hydroxylapatite-bound components (row 4) were eluted in phosphate buffer. PK: − and PK: +++, undetectable and strong protein kinase activities, respectively, assayed in vitro with NS1O as a substrate. F/T, flowthrough.