Figure 3
From: Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA
Detection of sequence-specific dsDNA binding to p65/RelA probe in the thermal shift assay. (a,b) The κB-dependent thermal stabilization of p651-306 probe in the presence of 10 µM dsDNA oligomers. Representative unfolding (left) and positive derivative (d(RFU)/dT) (right) curves are shown. Midpoint temperatures of the protein-unfolding transition (Tm) are presented as bars. Values are mean ± SD of at least three independent measurements (***p < 0.001). (c) EMSA of p651-306 protein (0.5 µM) using 32P-labelled 2κB and mutated κB consensus oligonucleotides (1 × 104 cpm). (d) Thermal stability shift assessed with varying 2κB dsDNA concentrations. ΔTm, change in Tm of p651-306 probe caused by dsDNA. The curve is based on two separate acquisitions shown as open and closed circles.
