FIG. 4.

Anti-RPA32 Ser-33, -RPA32 Ser-4/Ser-8, -Mre11, and -Rad52 antibodies immunoprecipitate the BMRF1 replication protein. Tet-BZLF1/B95-8 cells were cultured in the presence of 2 μg of doxycycline/ml and harvested at 24 h p.i. Nuclear extracts were prepared as described in Materials and Methods and incubated with nonspecific anti-rabbit or mouse IgGs as a control or with anti-RPA32 Ser-33-, anti-RPA32 Ser-4/Ser-8-, anti-BMRF1-, anti-Mre11-, or anti-Rad52-specific antibodies. Twenty percent of the nuclear extract used for IP reactions was loaded as an input control. Proteins from the immunoprecipitates were applied for SDS-PAGE, and the BMRF1 protein was detected by Western blotting with peroxidase-labeled anti-BMRF1 antibodies. The top and bottom show IP from nuclear extracts treated without and with DNase I, respectively. IB, immunoblot.