Fig. 1

Fig. 1

(a) Transmission pattern of GBP1 variations in three pedigrees. Proband 79 (CH 79) carried compound heterozygous variations (p.H150Y and p.E336fs) inherited from the euthyroid father and mother respectively. Proband 90 (CH 90) carried heterozygous variation inherited from the euthyroid mother. Proband 168 (CH 168) carried a heterozygous variation (p.L187P). V variation, black solid box: proband. (b) The cg12054698 methylation status examined in CH 90, CH 168, and their parents by pyrosequencing. F father, M mother. Relative methylation level of the CpG site of each sample was labeled. (c) The correlation of cg12054698 methylation status with the messenger RNA (mRNA) levels of human GBP1 (hGBP1) in 57 normal thyroid tissues (Spearman r = −0.61, P < 0.001). (d) Left is the quantification of GBP1 expression by immunohistochemistry (IHC) classified by cg12054698 methylation level assessed by methylation-specific polymerase chain reaction (PCR). Right is the representative IHC staining of GBP1 in cg12054698 high, moderate, and low methylated thyroid tissues. Bars = 50 μm. (e) Western blot examination of the R20X mutated GBP1 expression in TPC1 cells. (f) Western blot confirmation of the truncated GBP1 caused by p.E336fs variation in TPC1 cells. (g) The position of the two missense variations of hGBP1 (H150Y and L187P) in the secondary structure of hGBP1 protein. (h) The cellular location of transfected Flag-hGBP1, Flag-hGBP1-H150Y, Flag-hGBP1-L187P, and Flag-hGBP1-E336fs in TPC1 cells by immunofluorescence assay. Scale bar: 10 μm. (i) The expression of Flag-hGBP1, Flag-hGBP1-H150Y, and Flag-hGBP1-L187P in the cell membrane and cytosol compartment detected by western blot. Na/K⁺ ATPase was used as a loading control for the membrane and α-tubulin for the cytosol fraction. (j) The ratios of hGBP1 expression in the membrane to that in the cytosol, calculated by gray value scan in three independent experiments. (k) Membrane cytosol extraction show the content of the mutated hGBP1 (p.E336fs) in each compartment. Na/K⁺ ATPase was used as a loading control for the membrane and α-tubulin for the cytosol fraction. Means ± SEM are shown for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; (Student’s t-test).