(a, c, e) [¹²⁵I]T3 dissociation curves showing that compared with (a) WT, (c) the TRα1-M256T mutation, and (e) TRα1-M256A mutation reduces the affinity for T3 (solid line) more than for T4 (dashed line) (mean ± SEM of three experiments for WT and M256T and two experiments for M256A performed in duplicate). (b, d, f) The TRα1-M256T and TRα1-M256A mutations also had a larger effect on T3- than on T4-dependent transcriptional activation (mean ±SEM of three experiments performed in triplicate). The effect of the Ala substitution on the ligand binding affinity and the transcriptional activity of TRα1 was less than the effect of the Thr substitution. The insert in (b) shows immunoblots confirming an equal expression of WT, M256T, and M256A FLAG-tagged TRα1 and Histone 3 as a loading control in the nuclear fraction of JEG-3 cells.