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. 2014 Mar 13;63(4):1394–1409. doi: 10.2337/db13-0334

Figure 3.

Figure 3

4548-G05 binds to the ECD of IR. A: D4548-G05 alters the proteolytic cleavage pattern of IR-ECD. Recombinant IR-ECD (left panel) and human zyxin fragment (right panel) were subjected to limited trypsin digestion in the presence of DMSO or 4548-G05 (50 μmol/L). The reaction was resolved in SDS-PAGE followed by silver staining. B: Intrinsic fluorescence emission quenching of recombinant IR-ECD (250 nmol/L) with increasing concentrations of 4548-G05. C: Titration curve of IR-ECD fluorescence quenching in the presence of 4548-G05. D: 4548-G05 is a reversible ligand of IR. The Trp fluorescence of IR was decreased when 4548-G05 bound to IR (IR + G5). After centrifugation, the unbound 4548-G05 was filtered. When the 4548-G05/IR complex in the top chamber was reconstituted, some of the bound 4548-G05 dissociated from the ligand/receptor complex and reached a new equilibrium, which is indicated by the increase of intrinsic IR fluorescence (IR + G5 [centrifugation]). E: 4548-G05 does not compete with insulin for IR binding. CHO-IR cells were incubated with FITC-insulin in the presence of unlabeled insulin (left panel) or various concentration of 4548-G05 (middle panel). Parental CHO cells were used as a negative control (right panel). The fluorescence-labeled cells were analyzed by flow cytometry.