Fig. 3. Apparent Rad4-binding affinities of DNA constructs measured by competition electrophoretic mobility shift assays (EMSA). (A) Typical gel images showing the wild-type Rad4–Rad23 complex binding to various DNA constructs. ‘NPOM-DNA + hν’ indicates NPOM-DNA photocleaved by λ = 365 nm light applied for 3 min. Mismatched (CCC/CCC) and matched (CCC/GGG) DNA represent typical specific and nonspecific binding substrates, respectively. The sequences of DNA are in Table S1 (ESI†). (B) Quantification of the Rad4-bound DNA fractions versus total concentrations of the protein from gels including those shown in (B). The symbols and error bars indicate the means and ranges as calculated by ± sample standard deviations, respectively, from triplicate experiments. Solid lines indicate the fit curves of the data point. (C) K_d,app and R² of the fits derived from (B). The errors indicate the errors of the nonlinear regression fit.