Phosphorylation of Z serine residue 186 is not required for Z binding to methylated Rp ZRE-2. (A) EMSA was performed using bacterially expressed purified GST-Z and 32P-labeled oligonucleotide probes containing the AP-1 site in the BMRF1 promoter and either unmethylated or methylated (as indicated) 32P-labeled oligonucleotide probes containing ZRE-2. (B) EMSA was performed using in vitro-translated wild-type Z (Wt Z) and Z(S186D) (as shown) and 32P-labeled oligonucleotide probes containing the AP-1 site in the BMRF1 promoter and either unmethylated or methylated (as indicated) 32P-labeled oligonucleotide probes containing ZRE-2. (C) 293/EBV-ZKO cells (20) were transfected with either control vector (Vector) or a vector expressing wild-type Z (Wt Z) or Z(S186D). Immunoblots were performed on cell extracts 2 days after transfection with monoclonal antibody directed against Z, R, BMRF1, or β-actin, as indicated. +, present; −, absent; retic, rabbit reticulocyte lysate.