Fig. 5. Effect of serine mutations in the CRH2 domain on binding. (A) Leptin binding by the different serine mutants. The same method as in Fig. 3 was applied. (B) K values of the serine mutants. Also included are the R² values of the fitted hyperbola and the relative expression levels (R.E.), as determined by ELISA. Owing to the low expression levels, the K_d of the I501S and I501SF502S mutation was not determined. Since binding of leptin is severely reduced in the case of the double mutants, alkaline phosphatase values are far from reaching their plateau levels, resulting in an unreliable fit and very large confidence intervals.