Ab17795

Membrane protein-enriched samples from cultured cells were used for loading, as the iodide transporter normally functions within the cell membrane. After a PBS wash, the cells were harvested using an elution buffer (20 mmol/L HEPES, pH 7.6, 20 mmol/L NaCl, 0.5 mmol/L EDTA, 10% glycerol) and scraped into a chilled microtube. The cells were sonicated and centrifuged for 30 min at 10,000g at 4 °C, and the pellet was resuspended using a membrane-solubilizing buffer (elution buffer with 1% triton X-100) and incubated on ice for 30 min. After centrifugation, the supernatant was used as the membrane protein-enriched sample. The sample were separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to poly vinylidene fluoride membrane. These membranes were blocked for 30 min at room temperature with 0.5% skim milk in PBS containing 0.1% (v/v) Tween 20 (PBS-T) and then incubated with appropriately diluted rabbit anti-SLC26A7 (1:1000; BMP084, MBL), rabbit anti-SLC26A4 (1:500; ab98091, Abcam), mouse anti- SLC5A5 (1:1000; ab17795, Abcam), or mouse anti-FLAG antibodies (1:1000; F1840, Sigma-Aldrich) as primary antibodies. A rabbit anti-sodium potassium ATPase antibody (1:10000; ab76020, Abcam) and a mouse anti-β-actin (1:10000; A5441; Sigma-Aldrich) antibody were used as internal controls.

Standard IHC protocol was followed to stain the tumor tissue samples using the mouse monoclonal antibody against hNIS (human Sodium Iodide Symporter) (Abcam, ab17795), ER (Estrogen Receptor) (Abcam, ab16660, ab288). Briefly, 5 µm sized paraffin embedded tissue sections were de-paraffinized with xylene and endogenous peroxidase activity was quenched with 3% H₂O₂ in methanol for 30 minutes in the dark. Tissue sections were dehydrated through graded alcohols and subjected to antigen retrieval using 10mM sodium citrate. Sections were washed with TBST (Tris Borate Saline Tween-20) and then blocked with 5% BSA (Bovine Serum Albumin) for one hour. Slides were incubated with the respective mouse monoclonal primary antibody diluted with TBS. Slides were then washed for 5 minutes in TBST and incubated for 1 hour with the respective HRP (Horse Raddish Peroxidase) conjugated anti-mouse secondary antibody diluted with TBS in a ratio of 1∶200. After washing, slides were incubated with DAB (3,3′-diaminobenzidine tetrahydrochloride) (Sigma) and immediately washed under tap water after color development. Slides were then counter stained with hematoxylin. Slides were mounted with DPX (dibutyl phthalate xylene) and were then observed under a light microscope (Carl Zeiss).

Embryoid bodies were washed with PBS and fixed with 4% PFA for 20 min. Cells were incubated for 60 min at room temperature with blocking solution, consisting of 5% donkey serum and 0.1% Triton-X in PBS, and divided into 1.5-ml microtubes for staining. EBs were incubated with primary antibodies at 4°C overnight and then rinsed three times with blocking solution before applying secondary antibodies for 60 min at room temperature. Primary antibodies against Sox17, FoxA2, Pax8, and TTF-1, as well as negative control and secondary antibodies, employed the same materials and dilution as used for flow cytometry. To identify thyrocytes, thyroid-specific antibodies including mouse anti-human TSHR (ab6044 Abcam, 1:50), mouse anti-human NIS (ab17795 Abcam, 1:50), rabbit anti-human TG (ab156008 Abcam, 1:50), and normal mouse IgG (sc2025 Santa Cruz Biotechnology) was applied for the negative controls. EBs were washed with PBS, spread on the surface of a glass slide, and then mounted with GelMount™ (Biomeda). Finally, all of the immunostained samples were examined under confocal microscope (Olympus).