Biochemical properties of thyroid peroxidase (TPO) expressed in human breast and mammary-derived cell lines - Fig 1

The biochemical properties of TPO protein expressed in breast tissues (A and B) and breast-derived cell lines (C). (A) N-linked glycan content in TPO expressed in breast tissues. Total cell extract was digested with PNGase F, then subjected to 8% SDS-PAGE, followed by Western blotting, and probing with TPO-specific mAb 47 monoclonal antibody. Controls were processed under the same conditions as the samples except that no enzyme was added. One representative immunoblot out of at least three independent experiments is shown. (B) Enzymatic activity of TPO expressed in breast tissues. Tissue lysate was incubated with TPO-specific mAb A4, then protein A agarose was added to precipitate immune complexes. TPO-antibody complexes bound to agarose were incubated with luminol in the presence of hydrogen peroxide. The intensity of luminescencent signal was measured and results were expressed as relative light units (RLU). As positive control, TPO immunoprecipitated from human thyroid tissue lysate (Graves’ disease case) was used to measure luminol oxidation. Agarose A incubated with mAb A4 alone (lysate omitted) was used as negative control. One representative of three independent experiments is shown. (C) TPO protein expression in breast epithelial normal (184A1) and cancer cell lines (MCF-7 and MDA-MB-231). Western blotting was used to detect TPO protein presence. The specificity of the reaction was verified by preabsorption of ab76935 antibody with the excess of highly purified human TPO. NTHY was used as a positive control. β-actin-specific Ab was used as a loading control. BN: peri-tumoral breast tissue; BC: breast cancer tissue; G-B: Graves’ disease thyroid tissue; NTHY: NTHY-ori 3–1 cell line; PNGase F: Peptide-N-Glycosidase F; RLU: relative light units.