Fig. 5.

aDMA-modified EBNA2 is present in DNA-binding complexes. (A) EBNA2 and RBPJκ synthesized in a coupled in vitro transcription-translation system derived from rabbit reticulocytes (Promega) were used in a gel shift (EMSA) (Meitinger et al., 1994) after preincubation of the proteins with the indicated antibodies. The complexes III and IV which are formed by RBPJk and RBPJκ plus EBNA2, respectively, as well as complex IV supershifted with mAb R3 (lane 4) or aDMA-specific mAb 6F12 (lane 11) are indicated. (B) EBNA2-containing Raji cell extract was incubated with the indicated antibodies and then assayed in a gel shift assay. R3 recognizes EBNA2 regardless of its methylation status and induces a “supershift” indicated by the upper arrow (Zimber Strobl et al., 1993), the mAb 6C8 directed against the “WWP”-repeat of EBNA2 destroys the EBNA2/RBPJκ-complex IV (Sauder et al., 1994). Antibodies used are indicated above each lane. Control antibodies corresponded to the respective IgG-subtype of each antibody. To efficiently separate the high molecular weight complexes, the electrophoresis was carried out for an extended time. Therefore, uncomplexed ³²P-labelled probe ran out of the gel. The position of the RBPJκ-containing complexes I-IV as described in the text are indicated; the arrow points at the EBNA2-containing complex IV that is supershifted by R3 and aDMA-6F12 but destroyed by WWP-6C8.