FIG. 2.

Validation of BGLF4 and CDK1/cyclin B substrates. (A and B) In vitro kinase assays using GST-BGLF4 (A) or CDK1/cyclin B (B) and the indicated GST target proteins purified from yeast and incubated in kinase buffer containing [γ-³²P]ATP. Samples were subjected to gel electrophoretic separation, and the dried gels were exposed to X-ray film. Phosphorylated target proteins are indicated by asterisks. (C) Phosphorylation of EBNA1 (BKRF1) and TK (BXLF1) by BGLF4 in HeLa cells transfected with GFP-EBNA1 or V5-TK alone or together with Flag-BGLF4 and incubated for 2 h in medium containing [³²P]orthophosphate prior to being harvested. (Top) Phosphorylation of the EBNA.OT1x and anti-V5 immunoprecipitates visualized by autoradiography. (Bottom) Verification of GFP-EBNA1 and V5-TK expression by Western blotting of EBNA.OT1x and anti-V5 immunoprecipitates.