FIG. 2.

Specific interaction between pp71 and hDaxx as determined in coimmunoprecipitation experiments. 293 cells were transfected with eukaryotic expression vectors encoding myc-pp71, FLAG-hDaxx, and FLAG-hSPT6, as well as the HCMV protein ppUL69, as indicated and prepared for immunoprecipitation as described in Materials and Methods. Immunoprecipitation was performed with antibodies as indicated by bars. Precipitates were washed three times and separated by SDS-10% PAGE. (A) Immunoprecipitations were performed with the anti-FLAG monoclonal antibody or an anti-IE2 monoclonal antibody as indicated. Thereafter, coprecipitated pp71protein was detected in Western blot analysis with the anti-myc monoclonal antibody. Lanes: 1, lysates from untransfected cells; 2, transfection with plasmid myc-pp71 alone; 3, transfection with vector FLAG-hDaxx alone; 4 and 5, transfections with a combination of vectors encoding myc-pp71 and FLAG-hDaxx; 6, transfection with plasmid myc-pp71 and a plasmid encoding FLAG-Sp100. (B) Immunoprecipitations were performed with the anti-myc monoclonal antibody or the anti-IE2 monoclonal antibody. Interacting proteins were thereafter detected in a Western blot with the anti-FLAG monoclonal antibody. The lanes are analogous to those in panel A. (C) Immunoprecipitations were performed with the anti-UL69 monoclonal antibody; thereafter, coprecipitated proteins were detected by Western blot analysis with the anti-FLAG monoclonal antibody. The interaction between ppUL69 and the cellular protein hSPT6 served as a positive control (lane 2). Lanes: 1, transfection of expression vector pCB6-UL69; 2, transfection with a combination of plasmids encoding ppUL69 and FLAG-hSPT6; 3, transfection with plasmid FLAG-hDaxx; 4, transfection with constructs expressing FLAG-hDaxx and ppUL69. Molecular masses are indicated in kilodaltons. Abbreviations: IP, immunoprecipitation; wb, Western blot; IgG, immunoglobulin G.