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. 1999 Mar;19(3):2289–2299. doi: 10.1128/mcb.19.3.2289

FIG. 2.

FIG. 2

FIG. 2

Specificity of WT1–Dax-1 complexes. (A to D) WTZF(−KTS) recombinant protein was used in EMSAs with oligonucleotides containing the sequences corresponding to regions designated WB1 (A), WB1(m), a mutant of WB1 (B), WB2 (C), and WB2(m), a deletion mutant (D). The nucleotide sequences of the oligonucleotides are presented in Materials and Methods. The amount of recombinant WTZF protein used in the EMSAs was titrated and is indicated (in nanograms) above each lane. (E to H) Competition experiments were performed in the presence of increasing amounts (the molar excess is indicated above each lane) of an unlabeled oligonucleotide harboring either the WTE recognition site (E and G) or a mutant version of WTE (F and H). Specific complexes were preformed between WTZF(−KTS) protein and an oligonucleotide containing the WB1 site (E and F) or between WTZF(−KTS) protein and an oligonucleotide containing the WB2 site (G and H). Protein-DNA complexes were resolved on nondenaturing 4% polyacrylamide (acrylamide/bisacrylamide ratio, 37/1) gels electrophoresed at 4°C in 0.5× TBE. To detect the complexes, the gel was dried and exposed to X-Omat film (Kodak) at −70°C for 12 h. The positions of migration of free probe and protein-DNA complexes are indicated.