(A) Serum T4, T3, and TSH concentrations in WT and THRB K146Q mice (n = 13/ genotype) are shown as mean (±SD) and paired t test for statistical analysis. TSH is shown in log₁₀ scale because of the wide differences in the levels in WT and K146Q mice. (B) Thyroid was rinsed with saline, patted dry, and weighed. The weight is shown as wet weight per mouse (n = 18/genotype). (C) Representative histology of thyroid gland stained with H&E. Transverse section of thyroid gland (top panel) and thyroid follicles (lower panel). (D) Dissected pituitaries were rinsed with saline, patted dry, and weighed, and values are shown as wet weight of pituitary from each mouse (n = 13/genotype). (E) Pituitaries are shown from WT and THRB K146Q mice. (F) Image of representative pituitary tissue histology with H&E stain from WT and THRB K146Q mutant mice. (G) Immunofluorescence staining for TSHβ (green) and for nuclei (DAPI blue). Frozen sections of the pituitaries were incubated with anti-TSHβ antibody at 1:50 dilution and conjugated with Alexa Fluor 488. (H) The TSHβ-expressing cells and total cell numbers were counted using green and blue filters. (I) Western blot detection of TSHβ and common glycoprotein α subunit (CGα) proteins. Pituitaries (n = 3) were lysed in RIPA buffer, and 30 μg of protein was loaded on an 8% SDS gel. Membranes were Ponceau S–stained (Supplemental Figure 2) prior to blot with anti-TSHβ and anti-CGα. (J) Quantification of TSHβ and CGα protein band in Western blot using LI-COR Image Studio Lite. (K and L) Western blot detection of THRB protein in the thyroid and pituitary. Protein (30 μg) was loaded onto a 10% SDS gel, transferred to a PVDF membrane, and blotted with anti-THRB antibody. The protein loading is shown (Supplemental Figure 3). Statistical analysis was performed using paired t test (A, B, D, and H). THRB, thyroid hormone receptor β; K146, lysine 146; TSH, thyrotropin-stimulating hormone, T4, thyroxine; T3, triiodothyronine.