A. Diagram of the binding reaction between Ven-SH3 (Mona/Gads) and Cer-X, where X represents one of several binding partners of Mona/Gads. B. FRET binding curves for Ven-SH3 in complex with SLP-76, aa.231-243 (black), Gab1, aa.515-527 (green), USP8, aa.403-415 (blue), SLP-76 (D236K), aa.231-243 (cyan), and SLP-76 (K240R), aa.231-243 (red). Their respective binding affinities were Kd = 0.8, 1, 4, 7 and 24 μM, with Emax = 0.39, 0.4, 0.37, 0.35 and 0.35 respectively. C. Comparison between dissociation constants measured by flow cytometry (y-axis) and those measured by ITC (x-axis). On average, affinities measured by flow cytometry are ~3-fold less.