b>d determination
Saturation binding was performed with dilutions of [¹²⁵I]NKA to give a range of concentrations from 0.01 to 1.34 nM in the wells. Figure 1 shows the saturation curve, which was fitted using non-linear regression with the data analysis software package GraphPad Prism v4.0. A K_d value of 0.4 nM (95% confidence intervals 0.3–0.4 nM) was estimated directly from the curve.

IC₅₀ determination
Competitive binding of 0.17 nM [¹²⁵I]NKA with unlabeled NKA (4–10) was assessed and the IC₅₀ calculated as shown in Figure 2. Final concentrations in the well were 0.002–10 000 nM. Over two experiments, the mean NKA (4–10) IC₅₀ value was determined to be 18.9 nM (95% confidence interval range 13.7–26.1 nM) and mean K_i value was 13.3 nM (95% confidence intervals 9.6–18.3 nM).

Time course
In addition, a time course was performed using standard reagent concentrations detailed in the protocol with an increased incubation time of 18 h. The assay appeared to be stable for the full 18 h as shown in Figure 3.

Z’ analysis
Finally, a Z’ analysis was performed using 42 replicate values for "total" and NSB wells (6). The Z’ value indicated by Figure 4 is 0.62, which was well within the acceptable Z’ value range (0.5–1.0) and confirmed the robustness of the assay.

References
1. Patacchini, R. et al. Trends in Pharmacological Sciences 25, 1–3 (2004).

2. Longmore, J. et al. Canadian Journal Physiology and Pharmacology 75, 612–621 (1997).

3. Regoli, D. et al. Pharmacological
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