Electrophoretic Mobility Shift Assays using endothelial nuclear extracts derived from BOECs. Nuclear extracts from BOECs were tested with a 32P- labeled VWF WT probe spanning nucleotides −61 to −32 and a Mut probe spanning −70 to −32. (A) The pattern of DNA-protein complexes is distinct for the WT and Mut probes. Complexes are specific, because they disappeared in competition studies using 50× unlabeled probe. DNA-protein complexes with both the WT and mutant probes were “super-shifted” using anti–GATA-3 antibodies. (B) WT-DNA protein complexes from a repeated and independent experiment. The DNA-protein complex is “super-shifted” using anti-Ets antibodies (C) Mut DNA-protein complexes. The complex with the mutant probe is also “super-shifted” using anti-Ets antibodies. The images shown in panels B and C are from the same electrophoresis gel run. Repeated lanes have been removed to avoid confusion. (D) Cross-competition EMSAs. WT and Mut probes consistently show distinct banding patterns. Cross competition using cold WT and cold Mut oligos (10-, 50-, and 100-fold excess) shows that the cold WT probe competes both WT and Mut probe binding, whereas cold Mut competes only the Mut probe binding. Successful competition with WT probe binding is seen with the cold Mut oligo only at 100-fold excess. NE = nuclear extract. The vertical lines inserted between lanes in panels B and C indicate places where lanes from the same gel and experiment have been juxtaposed to clarify the data.
