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Fig. 3. Effect of mutations in the CRH2 domain on binding. (A) Leptin binding by selected mutants. Cos-1 cells were transiently transfected with pMET7-mLRCRH2-His6 plasmids encoding different polyhistidine-tagged CRH2 domain mutants. The collected supernatants were incubated in triplicate in pent-His antibody-coated plates. Binding of leptin was investigated by adding a serial dilution of a leptin-SEAP fusion protein and measuring the resulting alkaline phosphatase (AP) activity. Data were fitted to a hyperbola (corresponding to a one site binding curve) using GraphPad Prism; curves shown are results of these fits. Results shown are representative of three separate experiments. (B) K_d values of alanine mutants with effect on binding. Also included are the R² values of the fitted hyperbola. R.E.: relative expression levels, as determined by ELISA, expressed as a percentage of wild type.

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