Effect of mutations on STAT3 binding to pY1068 dodecapeptide. (A) Biotinylated phosphorylated (p) Y1068 peptide or biotinylated nonphosphorylated Y1068 peptide (control) were immobilized onto streptavidin agarose beads and incubated with lysates of STAT3^−/− MEF cells reconstituted with either WT or mutant STAT3 constructs. Bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with antibody against total STAT3. The red asterisk (∗) indicates that the images are a composite of 2 separate blots imaged simultaneously with equal exposure. (B) SPR analysis of binding of WT and mutant core STAT3 (127-722) proteins to pY1068 peptide. K_D values were determined by fitting data to a 1-site equilibrium–binding model and the results shown are the mean of 3 experiments. Each K_D value was divided by the WT result of that experiment to determine the relative K_D value and the mean relative K_D of the 3 experiments shown; the dash (−) indicates the relative K_D could not be calculated. NB, no binding detected.