Finally, gel shift assays were performed using the nuclear extract from KSHV-infected cells to verify the ability of Egr-1 to bind ORF50P3 and ORF50P8. As expected, there was no hindrance in the migration of ORF50P probes without the addition of cell lysate ( Fig. 1C; lanes 1 and 5) . However, the presence of the lysate in the samples resulted in the formation of protein:DNA complexes indicated by a band shift (Fig. 1C , lanes 2 and 6). Egr-1 binding to ORF50P was confirmed by incubating lysates with specific Abs and performing a supershift. Samples that were incubated with nonspecific IgG Abs displayed band shifts that were similar to samples containing only Egr-1 and the respective probes (Fig. 1C, lanes 3 and 7) . Alternatively, a supershift occurred exhibiting a discrete band when nuclear lysates were pre-treated with Egr-1 specific Abs (Fig. 1C, lanes 4 and 8). Taken together, these experiments provide support for the ability of Egr-1 to specifically bind to two separate locations on KSHV ORF50P. IVT-synthesized Egr-1 binds to ORF50P probes. EMSA studies were performed using IVT-synthesized Egr-1 products and DIGlabeled ORF50P probes (see Table 1 ). (B) Mutations in the putative Egr-1 binding domain inhibit Egr-1 binding. EMSA experiments were performed using wt ORF50P3 and ORF50P8 probes as well as corresponding probes displaying mutations in the suspected Egr-1 binding domain (ORF50P3m and ORF50P8m). (C) Nuclear lysates from KSHV-infected cells formed a complex with ORF50P probes. BCBL-1 cells were synchronized in S phase of cell cycle according to earlier protocols [28] , treated with 20 ng/ml TPA for 8 h, and lysed. Nuclear extracts containing Egr-1 proteins were used to perform EMSA studies using ORF50P3 and ORF50P8 probes. Specific Egr-1 binding was confirmed by performing supershifts using specific antibodies to Egr-1 (lanes 4 and 8) or nonspecific IgGs (lanes 3 and 7). The arrowhead indicates protein/ DNA complex formation. Specific antibody/protein/DNA supershifts are denoted by the asterisk. doi:10.1371/journal.pone.0033364.g001