A Novel Homozygous Mutation of Thyroid Peroxidase Gene Abolishes a Disulfide Bond Leading to Congenital Hypothyroidism

Figure 4

Functional analyses of the identified novel TPO mutation. Protein expression levels of TPO (WT or Mut) were assessed using Western blotting with a monoclonal anti-TPO antibody. (a) The upper bands denote TPO proteins (103 kD). Monoclonal anti-beta-actin antibodies were used as the loading control. The lower bands denote beta-actin proteins (42 kD). Western blots were performed in triplicate. (b) Each TPO expression signal was expressed in relative arbitrary unit after normalizing against beta-actin (TPO/beta-actin ± SEM). Wild-type TPO expression level was set to 1, and control TPO expression level was set to 0. The mutant TPO expression level was significantly lower than that of the wild type (n = 3, Student’s t test, ). (c) The results of quantitative PCR performed using SYBR Green were shown in arbitrary units of TPO mRNA/housekeeping gene hprt1 as the mean ± standard error of the mean. There was no significant difference between wild-type and mutant TPO mRNA expression (n = 5, Student’s t test, ). Measurement of peroxidase activity using Amplex Red reagent. (d) Peroxidase activity of mutant TPO protein (Mut) was normalized to that of the wild type (WT; 100%) and that of the mock-transfected (control; 0%). The results of three independent experiments are expressed as the mean ± standard error of the mean. , Student’s t-test (Mut vs WT).

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