FIG. 3.

Serine phosphorylation of EBNA2 at the CKII site. 293T cells were transfected by the calcium phosphate technique with pSG5-FLAG-EBNA2 wild type or the SS469AA substitution mutant. Cells were lysed and processed as described in the legend to Fig. 2 in NP-40 lysis buffer supplemented with both protease inhibitor (Roche Diagnostics GmbH, Mannheim, Germany) and phosphatase inhibitor (phosphatase inhibitor cocktail I, P-2850, and phosphatase inhibitor cocktail II, P-5726 [Sigma]). Lysed cell supernatants were immunoprecipitated with anti-FLAG M2 affinity gel (Sigma), washed extensively, blotted, and probed with mouse monoclonal anti-phosphoserine ascites fluid, P-3430, (Sigma), or a rat monoclonal antibody against EBNA2 (R3). Immunoblots were developed by using ECL, and bands were quantitated by using OptiQuant version 4.0 analysis software (Packard Instrument Co.).