FIG. 2.

(A) Representative chromatograms of sequences from family members WT (c.653G, p.R218), heterozygous (c.653G>A, p.R218H monoallelic substitution), and homozygous (c.653G>A, p.R218H biallelic substitution) for the HSA mutation. (B) Scatter and box plots of total T4 concentrations in WT, heterozygous, and homozygous individuals. Line represents the median, and whiskers represent the range, when applicable. (C) Quantification of relative T4 content in bands representing TBG, HSA, and TTR separated by non-denaturing PAGE shown in Figure 1C. The table below the graph shows the corresponding measured concentrations of T4, TBG, and HSA in sera from these individuals, and calculated quantity of T4 bound to HSA and TBG, adjusted for the serum concentrations of these proteins. PAGE was carried out as follows: 1 μL containing 20,000 counts per minute (cpm; or 16 pg) of ¹²⁵I-labeled T4 (specific activity 1250 μCi/μg) was added to 10 μL of serum from each individual and incubated for 30 min at room temperature. The added ¹²⁵I-T4 thus represented 0.6–2.2% the endogenous T4 measured in the serum samples. Following dilution in buffer (250 mM of Tris•Cl, pH 8.5), 6 μL was loaded for separation by 8% non-denaturing PAGE at 60 V for 4 h at room temperature. The gel was subsequently exposed to film at −80°C with an intensifying screen for 1–4 days and scanned into an electronic format, and relative quantification of the bands was performed using the publically available NIH ImageJ software.