Hotspot Peptide Ligand Kcf12 Competes TNF Binding To Endogenous TNFR2 Receptors. The data are from representative TNFα and KcF12 competition experiments in Jurkat and Jiyoye cells. The binding of TNFα to TNFR2 on intact cells was measured in a radio-receptor assay using Jurkat and Jiyoye cells and the MultiSreen™ assay filter plate system as described . Data analysis was performed with an add-in for Microsoft Excel, Xlfit™ (ID Business Solutions, Guildford, UK), which allows curve fitting using nonlinear regression. To determine binding kinetic parameters, the four-parameter logistic equation y = Bottom + (Top – Bottom)/(1 + (IC50/x)Hill slope) was used to calculate IC50 values and Hill slopes. Ki values were calculated according to the equation, Ki = IC50/(1 + [ligand]/Kd). Data from replicate experiments in Jurkat cells (upper panel) yielded IC50 value of 1.64 ± 0.62 × 10-9 M (mean ± SD, n = 4) with a Hill slope of -0.66 ± 0.07 for TNF-alpha. Data from replicate experiments in Jiyoye cells (lower panel) yielded IC50 value of 2.28 ± 0.96 × 10-10 M (mean ± SD, n = 4) with a Hill slope of -0.52 ± 0.03 for TNF-alpha. Data from replicate experiments in Jiyoye cells (lower panel) yielded IC50 values of 3.56 ± 0.35 × 10-6 M (mean ± SD, n = 4) for HotSpot TNFR2 peptide KcF12.