Figure 5

Transactivation potential of EWS (NTD)^B. (A) Schematic representation of the GAL4-fusion expression plasmids used in this study. The expression vectors driving the production of GAL4-EWS (NTD) or GAL4-EWS (NTD)^B are shown. GAL4, GAL4 DNA-binding domain; EWS (NTD), EWS N-terminal domain of EWS-Oct-4; EWS (NTD)^B, EWS N-terminal domain of EWS-Oct-4B. (B) Comparison of transactivation potential of EWS (NTD)^B. The reporter plasmid, 5 × Gal4 TATA luc, was cotransfected with GAL4-EWS (NTD) or GAL4-EWS (NTD)^B into 293T cells (top panel). Luciferase activity was expressed as fold activation relative to the basal level observed with the reporter plasmid and the GAL4 DNA-binding domain alone (lane 1). Each transfection was performed independently at least thrice and the mean values are plotted with their standard errors (±s.e., vertical bars). Extracts for luciferase assays were resolved using 12% SDS–PAGE, transferred to a PVDF membrane, and immunoblotted with anti-GAL-4 (RK5C1) (middle panel) or anti-EGFP (bottom panel) antibodies as indicated.