FIG. 2.

In vivo interactions of mutant EBNA2 and hSNF5/Ini1 as shown by coimmunoprecipitation of EBNA2 and FLAG-Ini1 in 293T cells. 293T cells were transfected by the calcium phosphate technique with 5 μg of indicated plasmid DNA. After 48 h, transfected cells were lysed in NP-40 lysis buffer (0.5% NP-40, 50 mM Tris [pH 7.5], 150 mM NaCl, 5 mM EDTA), the nuclear debris was removed by centrifugation, and the supernatant was used for immunoprecipitation by binding to anti-FLAG agarose (Sigma, Saint Louis, Mo.). (A) Samples were separated by SDS-PAGE, blotted, probed with the EBNA2-specific rat monoclonal antibody (R3) (14), and developed with anti-rat horseradish peroxidase and the ECL substrate kit (Amersham). Panels B and C are immunoblots showing the expression of EBNA2 and FLAG-Ini1, respectively. Panel D shows a coimmunoprecipitation experiment using a mutant of EBNA2, WW325FF, that does not bind to CBF1/RBPJκ.