A second set of EMSA studies were conducted using ORF50P probes carrying mutations (ORF50P3m and ORF50P8m; Table 1 ) in the putative Egr-1 binding region to further confirm the binding ability of Egr-1 proteins. Briefly, ORF50P3 and ORF50P8 were mutated to carry the 5 bp ATATA sequence in the GC-rich binding domain and then incubated in binding buffer alone or with IVT-synthesized Egr-1. Samples consisting of wildtype (wt) or mutated probes alone did not display a shift in the respective ORF50P3 and ORF50P8 probes ( Fig. 1B ; lanes 1, 3, 5. and 7). Following incubation of wt probes with Egr-1, complexes were formed producing separate band shifts (Fig. 1B , lane 2 and 6). Interestingly, Egr-1 did not bind ORF50P3m or ORF50P8m probes that displayed mutations in the Egr-1 binding domain, thus confirming the necessity for the consensus GC-rich binding domain mediating Egr-1/ORF50P interactions (Fig. 1B, lanes 4  and 8) .