Fig. 3. Binding affinity of Snail, E47 and Slug recombinant proteins to E-pal synthetic oligonucleotide estimated by capillary electrophoretic mobility shift assays (CEMSA). (A,B) Electrophoregrams for mixtures of 6-FAM-labeled wild-type E-pal oligonucleotide (24 nM) and (A) 1 nM of control GST, GST-Snail, GST-E47 and GST-Slug or (B) 1 µM of GST-Snail, GST-E47 and GST-Slug, subjected to CEMSA analysis as described in Materials and Methods. RFU, relative fluorescence units; min, elution time in minutes. The elution of the main E-pal oligo—GST-Snail complex is indicated by arrows (E-pal/protein) in both panels. (C) Concentration-dependent binding of GST-Snail (white circles), GST-E47 (black circles) and GST-Slug (white squares) to the 6-FAM wild-type E-pal probe. The indicated concentrations of the different recombinant proteins were analyzed by CEMSA, in duplicated samples, in three independent experiments. The single-site ligand-binding fit was performed using GraFit 3.1 software. R, saturation ([complex]/[complex]+[DNA]). [protein], protein concentration of intact GST-factors. Results are expressed as mean±s.d. (D) Western blot analysis of purified recombinant proteins. 50 µg of the purified recombinant proteins analyzed with anti-GST or anti-E47 polyclonal antibodies, as indicated. Migration of the molecular weight markers (in kDa) and of the different intact recombinant proteins is indicated at the side of the panel.

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