Assay development and stability assessment.

A. Unlabeled peptide 1 (Ac-PFKPP-NH2) and mutant peptide 2 (Ac-PAKPP-NH2) were titrated into a mixture of c-Cbl (RING) protein (3 µmol/L)+100 nmol/L probe 1 separately in the detergent-free system. IC50 (peptide 1) = 1.05±0.09 µmol/L. B. Unlabeled peptide 1 and mutant peptide 2 were titrated into a mixture of c-Cbl (RING) protein (3 µmol/L)+100 nmol/L probe 1 separately in the detergent-present system. IC50 (peptide 1) = 1.06±0.09 µmol/L. C and D, temperature dependence of Kd and ΔmP values in detergent-free or detergent-present system. c-Cbl (RING) protein was titrated against 100 nmol/L probe 1. E. Assessment of stability of binding experiments over time in detergent-free or detergent-present system. c-Cbl (RING) protein was titrated against 100 nmol/L probe 1. FP values were measured at 25°C up to 180 min. F. Stability of binding experiments with increasing DMSO concentrations in detergent-free or detergent-present system. DMSO was titrated into constant amounts of c-Cbl (RING) protein (3 µmol/L) and probe 1 (100 nmol/L) mixture. The total reaction volume was maintained constant in all wells at 20 µl. The measurements were made at room temperature (25°C) after 30 min of incubation.