(A and B) Single cell data show decreasing receptor availability and increasing ligand availability with increasing ligand expression. Circles denote the medians of data points in logarithmically spaced bins along the x-axis. ‘Effective total ligand’ refers to the ligand availability observed at a given ligand-CFP fluorescence value in a cell line expressing only ligand. For receptor availability data in A, n = 299 and in B, n = 352. For the ligand availability data in A, n = 323 and in B, n = 530. Gray bars in all panels represent background levels, defined as the 25–75 percentile range of fluorescence from parental CHO-K1 cells that do not express Notch1 or ligand-Cerulean constructs. (C) Transient expression of wild-type Dll1-mCherry (n = 8817), but not the Dll1-mCherry F199A mutant (n = 14,292), reduced Notch availability to background levels in a Notch1 cell line. Cells were analyzed by flow cytometry. Error bars in all panels denote 95% confidence interval for the bootstrapped estimate of the median. (D) Comparison of Notch availability in the Notch1+Dll1 and Notch1+Jag1 cell lines. Lines are fits to a model of receptor-ligand cis-interactions (Supplementary). (E) Comparison of ligand availability in cell lines expressing Dll1. Ligand availability a cell line expressing Dll1 only (n = 1146). Notch1 reduces ligand availability (purple, n = 1131), and this effect is rescued by siRNA against Notch1 (orange, n = 972). In the purple starred region, cells differ significantly in ligand availability between Notch1 or no target siRNA samples, while the orange star denotes regions where Dll1 and Notch1+Dll1 cells transfected with no target siRNA differ significantly. Significance was determined by applying the Wilcoxon rank sum test. Inset shows the model behavior for parameters derived from the fit in D. Knockdown of Notch was measured to be 50%. (F) Comparisons of ligand availability in a cell line expressing Jag1 only (green, n = 733), Notch1+Jag1 (orange, n = 532), and Notch1+Jag1 with siRNA against Notch (purple, n = 1163). Starred regions indicate significance as in E. Inset shows model behavior using parameters measured in D. Knockdown of Notch was measured to be 70%.