Extended Data Fig. 5: EMSA off-rate analysis at non-consensus NF-κB motifs.
From: Stepwise neofunctionalization of the NF-κB family member Rel during vertebrate evolution
a, EMSA off-rate profiles are shown for Rel and RelA RHRs from representative species (human, mouse, and zebrafish) expressed in HEK293T cells. Profiles are shown for experiments performed with oligonucleotides containing the non-consensus Il12b κB2 motif (middle) and another non-consensus motif from the Il2 locus (CD28 response element, right)53. Complex abundances (y-axis) are presented as the mean percentage (complex abundance at the 0-min time point set at 100%) at each time point (x-axis). Standard deviations are included when results represented the mean of three experiments, but not when the results represented the mean of two experiments. At the left, the profiles obtained with a consensus motif are reproduced from Fig. 6b for comparison. b, EMSA off-rate experiments are shown for Rel RHR mutant proteins expressed in HEK 293 T cells bound to the non-consensus Il12b κB2 motif. At the left, representative gel images are shown from two independent experiments. Note the appearance of a non-specific band (ns) that remains stable at all time points with the mutant proteins. Also note that the specific band is difficult to detect at the 0 min time point with the R101A mutant, due to the low affinity of the mutant protein for the non-consensus sequence. At the right, the EMSA off-rate profiles are shown for the Rel mutant proteins with the probe containing the Il12b κB2 motif. Complex abundances (y-axis) are presented as the mean percentage (complex abundance at the 0-min time point set at 100%) from two independent experiments at each time point (x-axis). Data from only one experiment for the R101A mutation are presented because the complex at the 0 min time point was too weak to quantify in other replicates).
