Figure 2

The UtroUp DNA-binding affinity/specificity analysed by EMSA. A: An equal amount of the GST-UtroUp protein (14 nM) was incubated with an equal amount of either the labelled wild-type DNA target (lanes 1–2) or with the labelled scrambled DNA target (lanes 3–4). B: (Left) An increasing amount of the GST-UtroUp protein (20, 40, 80 nM) was incubated with an equal amount of either the labelled wild-type DNA target (lanes 1–4) or with the labelled mutant DNA target (lanes 5–8). The two mutated triplets in the DNA target are indicated by lowercase characters. (Right) Histogram shows a percentage of probes (WT and Mutated) shifted with increasing amounts of GST-UtroUp protein. C: An equal amount (12 nM), of the GST-UtroUp protein (lane 2) and GST-Jazz protein (lane 1) were incubated with an equal amount of labelled UtroUp-Jazz DNA target. D: An EMSA prototype performed for the determination of the dissociation constant (Kd). An increasing amount of GST-UtroUp protein (1, 2, 3, 4, 8, 12, 30, and 60 nM) was complexed with the labelled UtroUp DNA target. E: (Top) Quality control by Coomassie blue staining of purified/eluted GST-UtroUp and GST-Jazz fusion proteins compared with purified commercial bovine serum albumin. (Bottom) The Kd values of the GST-UtroUp/DNA-target and GST-Jazz/DNA complexes correspond approximately to 3,5 nM and 32 nM respectively. The protein concentration is expressed in nM.