FIGURE 2.

Real-time SPR measurement of the affinity of hPRL and its affinity matured variant hPRL^FulloPL toward rat PRLR-ECD. Left panels: the rat PRLR-ECD was immobilized on a nickel-nitrilotriacetic acid surface, then the hormone (350 nm) (a), buffer (dissociation phase) (b) and rPRLR-ECD (concentrations as indicated) (c) were successively injected onto the surface to measure site 1 (a) and sites 2+3 (c) interactions. The top panels correspond to hPRL^WT, and the bottom panels to hPRL^FulloPL. Right panels: sites 2+3 binding sensorgrams corresponding to injections of various concentrations of rPRLR-ECD onto pre-formed 1:1 ECD·PRL complexes, involving either hPRL^WT (top) or hPRL^FulloPL (see supplemental Fig. S2 for other hPRL variants), after subtraction of control sensorgrams corresponding to injections of buffer instead of rPRLR-ECD.