Effect of mutations on thermal stability of STAT3 protein monomers and homodimers. (A) Bacterially expressed and purified WT and mutant STAT3 proteins were examined for thermal stability using the differential scanning fluorimetry assay; change in fluorescence (dF) is recorded with increasing temperature (dT). The first derivative of each melting curve (dF/dT) is shown for WT and mutants organized into mutant categories and examined as monomers (right panels) or as homodimers (left panels). The inflection point or T_m for WT STAT3 monomers and homodimers is indicated by the vertical dashed line in each panel. (B-D) The calculated T_m of each mutant monomer, the primary T_m (T_m1) and secondary T_m (T_m2) of each GOF SH2D mutant homodimer, and the differences between the T_m, T_m1, or T_m2 of each mutant and the corresponding T_m of WT STAT3 (ΔT_m) are shown in tabular form (B) and graphically (C-D).