FIG. 2.

Construction and analyses of the FLAG-US28/1-314 HCMV recombinant in infected cells. (A) FIX-BAC DNAs isolated from E. coli were used as templates in all PCRs. Primers with homology to 5′ and 3′ sequences flanking the US28 coding region were used to PCR amplify the US28 locus (upper panel). The primer with homology to the 5′ flanking sequence was used in combination with a FLAG-specific primer to verify the addition of an N-terminal FLAG epitope (middle panel). Amplification of the UL146 gene serves as a positive PCR control (lower panel). (B) Immunoprecipitation followed by Western blotting with FLAG-specific antibodies was performed to detect expression of the various forms of FLAG-US28 encoded by HCMV ΔUS28, HCMV FLAG-US28/WT, and HCMV FLAG-US28/1-314 viruses at 48 h postinfection (upper panel). Whole-cell lysates from the same samples were subjected to Western blotting with an α-IE1/IE2 or α-UL44 antibodies (lower panels). The results shown are representative of six independent experiments.