Pierce protein biology

Hydrogen tetrachloroaurate-(III) trihydrate (HAuCl₄ ∙ 3H₂O, 99.99%), sodium borohydride (NaBH₄), Hexadecyltrimethylammonium bromide (CTAB, 96%), Cetyltrimethylammonium chloride solution (CTAC), ascorbic acid (AA) and Whatman^® qualitative filter paper, Grade 1 (Whatman no. 1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The synthetic RRWHRWWRR-NH₂ polypeptide (further noted as P2) was synthetized by Pierce Protein Biology, Thermo Fisher Scientific (Mt Prospect, IL, USA). All chemicals were of analytical grade, and all aqueous solutions were prepared using ultrapure water (resistivity~18 MΩ).

Cells were plated in 24-well plates (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h, treated with tunicamycin (Sigma, St. Louis, MO, USA) as a 10 mM stock solution in dimethyl sulfoxide, and stored at 36 °C for 24 h. After aspiration of the drug-containing medium, the cells were washed with 1 mL of Hanks balanced salt solution (HBSS) (Thermo Fisher Scientific) and incubated with 500 mL of HBSS containing 0.5% bovine serum albumin (Sigma) (bHBSS), 3.7 kBq of carrier-free ¹²⁵I (PerkinElmer, MA, USA), and a 10 µmol/L solution of sodium iodide (specific activity of 740 MBq/mmol) at 37 °C for 30 min. The cells were then washed twice with ice-cold bHBSS and lysed with 500 mL of 2% sodium dodecyl sulfate (Sigma). The radioactivity was measured using a gamma counter (Cobra II; Canberra Packard, Packard Bioscience; PerkinElmer). The radioactivity of the cells was normalized using total protein concentrations determined by a bicinchoninic acid protein assay kit (Pierce Protein Biology; Thermo Fisher Scientific). Some cells were preincubated with 300 μM KClO₄ (a specific inhibitor for NIS) (Sigma) for 30 min to inhibit iodide uptake, followed by the ¹²⁵I uptake test.