E EMSA titration experiment, with increasing concentrations of either Myc bHLH-LZ variant (WT or HEA) in presence of a fixed amount of the Max bHLH-LZ (2 μM) and of the fluorescently-labeled IRD-CACGTG probe (500 nM). The plot shows the mean and standard deviation of the quantification of the shifted Myc/Max bands as a function of the Myc concentration (the experiment was performed in triplicate). % Bound probe: fraction of the IRD-CACGTG probe associated with the Myc/Max complex. HEA/WT Kd ratio: Kd value for MycHEA/Max/CACGTG divided by the Kd value for MycWT/Max/CACGTG. The apparent Kd values were 3.78 μM for MycWT/Max and 5.57 μM for MycHEA/Max. Data information: Note that the differences in apparent Kd values between panels E and F-H can be explained by the different thresholds required to detect quantifiable complexes in the protein titration experiment (E), as opposed to a loss of signal intensity from an already detectable complex in the presence of unlabeled competitor (F-H): as such, competition experiments provide a better approximation of actual binding affinities. (F-H): The plots show the mean and standard deviation. The experiments were performed in triplicate. Unpaired Student t-test was used to compare IC50 values and expressed as p-values. 