FIG. 4.

EBNA-LP is associated with and phosphorylated by DNA-PK. (A) DNA-PKcs phosphorylation of GST-p53 is used to assay EBNA-LP-associated DNA-PKcs activity. FLP adsorbed onto M2 beads from lysates of FLP-expressing BJAB cells was incubated in kinase buffer with purified recombinant GST-p53 (100 ng) or control protein and [γ-³²P]ATP. ³²P-labeled GST-p53 was analyzed for phosphorylation level by PAGE followed by exposure to X-ray film (Auto). Coomassie brilliant blue (Coom) was used to estimate FLP abundance. (B) FLP adsorbed onto on M2 beads from BFLP lysates was incubated with [γ-³²P]ATP under in vitro kinase assay conditions in the presence (+Wort) or absence of 50 μM Wortmannin, a specific in vitro inhibitor of DNA-PK, or with 5 μM PKA or PKC inhibitory peptides (PKAin or PKCin, respectively). (Sigma-Aldrich). Coomassie brilliant blue is used to estimate protein levels, and phosphorylation was estimated by autoradiography.