The contribution of NSB to total binding of homologous competition data depends on the concentration of cold EB when ligand depletion is significant. A. The presence of NSB (α = 0.1, [3H]EB = 0.013 nM) displaces the baseline of bound [3H]EB above the x-axis. Ligand depletion does not affect the size of this signal at large concentrations of cold EB, which in this case is equivalent to the apparent NSB. As anticipated from Figure 7, increasing ligand depletion in the presence of NSB shifts IC50 rightward and distorts the sigmoidal shape of the competition curve. R1T values increase in one log unit increments. B. NSB is not constant along the x-axis. Increasing ligand depletion with increasing R1T reveals the sigmoidal or distorted sigmoidal appearance of NSB as a function of cold EB and shifts the NSB curve rightward. Conditions and legend are the same as in A.