Egr-1 binds at least two different sites within the ORF50P Egr-1 is said to bind a GC-rich DNA template (such as GCGC(G/T)GGGCG, GCGGGGGCG, and CGCCCATGC) on the promoter and initiate gene transcription [15, 16] . Eight possible GC-rich Egr-1 binding sequences have been identified by us in the promoter region of KSHV ORF50. In order to determine the sites where Egr-1 bound ORF50P, EMSA experiments were performed using 8 different DIG-labeled probes, referring to identified locations on ORF50P, (Table 1) and Egr-1 in vitro transcribed and translated (IVT) proteins. IVT of egr-1/ pcDNA3.1(+) construct yielded a protein of roughly 78 kda (data not shown) [6] . Of all the probes tested, IVT-synthesized Egr-1 proteins were able to bind and form separate protein:DNA complexes with the ORF50P3 and ORF50P8 probes, respectively; displaying distinct band shifts when compared to controls using the probes alone ( Fig. 1A ; lanes 6 and 16). It is important to note that the sequence for ORF50P3 is the same for the probe used in earlier study [6] . Band shifts were not observed when ORF50P probes were incubated with IVT-synthesized KSHV glycoprotein L (gL) (data not shown). Additionally, experiments using the ORF50PNP probe (does not contain the GC-rich binding domain) did not form a complex with Egr-1 protein, thus confirming the specificity of Egr-1 binding (Fig. 1A, lane 18) . Finally, competition experiments using unlabeled ORF50P probes reduced band shifts by preventing Egr-1 binding to DIG-labeled probes (data not shown).