Figure 3

Gel shift assay for nuclear protein binding on MYB2 site The analysis of the DNA-protein complex by gel shift assay was performed with nuclear extracts using 10 nM DIG-labeled probe fragment with nuclear proteins extracted from immature seed coats from flax (developmental stage 3). The gel shift assay was performed in a 6% polyacrylamide gel. White arrowheads show the retarded DNA signals. This Figure is representative of at least 5 independent experiments performed in the same conditions. a. Comparative analysis of the DNA-protein complex formation by gel shift assay performed using 10 nM DIG-labeled probe fragment with 10 μg of nuclear protein extracted with the different nuclear protein extraction methods. b. Effect of nuclear proteins (obtained with the optimized protocol) concentrations on DNA-binding capacity on the putative MYB2 site. The amount of nuclear proteins added per lane is indicated. c. Titration of nuclear proteins (obtained with the optimized protocol) specific affinity for the Dig-labeled MYB2 containing fragment using the same unlabelled fragment at the indicated fold-molar-excess in the presence of 10 μg nuclear proteins extract from immature flax seed coat. d. Effect of mutation of the putative MYB2 binding site on DNA-binding capacity of 10 μg nuclear proteins (obtained with the optimized protocol).