FIGURE 4.

Effect of various dCTP concentrations on inhibition with INDOPY-1. A, single nucleotide incorporation was monitored in the presence of increasing concentrations of INDOPY-1 (ranging from 0 to 30 μm) where INDOPY-1-mediated inhibition is indicated by the disappearance of the n+1 product band. The experiment was performed in the presence of either 0.15 μm dCTP or 0.5 μm dCTP, respectively. Lane 1 shows the pre-RT heparin addition control. B, logarithmic graph represents normalized percentage of product formed plotted as a function of INDOPY-1 concentration under two separate conditions (variable dCTP concentrations). We obtained an average IC₅₀ value of 6.5 ± 4 of three independent experiments at concentrations of 0.15 μm dCTP. IC₅₀ values cannot be accurately determined for higher substrate concentrations because the reaction is not efficiently blocked under these conditions. Saturating concentrations of ∼20 μm of INDOPY-1 can further limit the accuracy of these measurements.