Figure 4
DNA-binding property of the EWS-Oct-4B chimera. (A) Immunoblot analysis of EWS-Oct-4B and EWS-Oct-4 to quantify GST fusion proteins. The GST-EWS-Oct-4 fusion proteins used in EMSAs were fractionated on 10% SDS–PAGE and visualised by western blotting with an anti-GST antibody (B-14, Santa Cruz Biotechnology). (B) EMSAs of the DNA-binding properties of EWS-Oct-4B and EWS-Oct-4. The EMSAs were performed using recombinant GST (lane 1, 0.15 μg; lane 2, 0.45 μg; lane 3, 1.35 μg), GST-EWS-Oct-4 (lane 4, 0.15 μg; lane 5, 0.45 μg; lane 6; 1.35 μg), or GST-EWS-Oct-4B (lane 7, 0.15 μg; lane 8, 0.45 μg; lane 9, 1.35 μg), and radiolabeled probe, as described in Materials and Methods. The recombinant proteins used in each EMSA are indicated above the gel. Protein–DNA complexes were resolved on non-denaturing 4% polyacrylamide (acrylamide:bisacrylamide ratio, 37 : 1) gels run at 4°C in 0.5 × TBE (44.5 mM Tris-HCl, 44.5 mM boric acid, 1 mM EDTA). The positions of free probe and protein–DNA complexes are indicated.
