Ha tag antibody

Non-tissue culture-treated 24-well plates were coated overnight at 4°C with recombinant fibronectin fragment (RetroNectin; Takara) at 25 µg/mL in PBS. The following day, the RetroNection solution was replaced with 0.5 mL of sterile 2% BSA in PBS to block the plates for 30 min at room temperature. After a single wash with PBS, virus particles were added and then pelleted by centrifugation at 2,000 g for 2 hours at 32°C. The supernatant was gently removed, and the wells are washed with 500 µL PBS followed by adding 0.5 million T cells in 2 mL T cell medium supplemented with 10 µg/mL protamine sulfate. Next the plates were centrifuged at 500 g for 3 min and then incubated for 3 days. On the third day after transduction, the cells were pooled together, stained with HA-tag antibody (#901509, Biolegend), and sorted to purify TCR transduced population. After sorting, transduced T cells were cultured in IL-2 containing media (30 IU/mL) and split as necessary to maintain cell density between 0.5 and 4 million cells/mL.

The surface expression levels of NCC and its variants were analyzed by staining cells using anti-hemagglutinin (HA) tag antibody (catalog number 682404; BioLegend). An HA epitope with flanking “SGSGG” sequence on both sides was inserted into extracellular loop 4 of NCC between residues 414 and 415. The insertion of the HA epitope does not affect the transport activity or biochemical behavior of NCC, as evaluated by a radioactive iodide uptake assay and fluorescence-detection size-exclusion chromatography (FSEC) analysis, respectively. HEK293S cells were cotransfected with HA-tagged NCC variants and a yellow fluorescent protein (YFP), with the latter serving as a marker for successful transfection. After 24 h, cells were harvested and washed with ambient phosphate buffered saline (PBS) once. 0.5 × 10⁶ cells were stained by incubation in PBS with 4 μg/mL Alexa Fluor 647-conjugated anti-HA antibody (catalog number 682404; BioLegend) for 30 min at room temperature. Cells were then washed with ice-cold PBS (x 3 times) and loaded onto the flow cytometer (BD Accuri C6 Plus) for analysis (Supplementary Fig. 2). Cells expressing YFP (FITC positive) were used for measuring surface expression, and surface-expression levels of NCC variants were quantified by the mean fluorescence intensities of Alexa 647.