Figure 2. Relative binding affinities of AtMYB61 to CASTing targets and to mutated ACCTAC motif determined by nitrocellulose filter-binding assays are confirmed by electrophoretic mobility-shift assays (EMSAs).

(A) EMSA of recombinant AtMYB61 protein binding to 6 labelled CASTing target sequences. The protein concentration used was 5×10–08M. Protein concentrations were conducted at 5×10–08M because this was the protein concentration at which targets had not all reached their binding max as determined by nitrocellulose filter-binding assay, allowing one to observe differential binding. (B) EMSA validating relative binding affinities of AtMYB61 to mutated ACCTAC motif. The protein concentration used was 5×10–08M. Mutations were conducted by substituting a single guanine nucleotide along the AC1 element. Black arrow indicates gel shift by the probe. Non-binding site (NBS) is a sequence that does not bind AtMYB61, acting as a negative control. Probes were engineered for the EMSA reaction by inserting the hexamer CASTing sequence or mutated AC1 element sequence into the underlined area.