Carrier free 125i

Cells were plated in 24-well plates for 24 h and then treated with GSK5182 synthesized by Daegu-Gyeongbuk Medical Innovation Foundation and prepared as a 100 mM stock solution in dimethyl sulfoxide and stored at 280°C for 24 h. After aspiration of drugcontaining medium, the cells were washed with 1 mL of Hanks balanced salt solution (HBSS) and incubated with 500 mL of HBSS containing 0.5% bovine serum albumin (bHBSS), 3.7 kBq of carrierfree 125 I (Perkin-Elmer), and a 10 mmol/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 were lysed with 500 mL of 2% sodium dodecyl sulfate. The radioactivity was measured using a g counter (Cobra II; Canberra Packard, Packard Bioscience). The radioactivity of the cells was normalized using total protein concentrations determined by a bicinchoninic acid protein assay kit (Pierce Protein Biology). The cells were preincubated with 300 mM KClO 4 (as a specific inhibitor for NIS) for 30 min to inhibit iodide uptake, followed by a 125 I uptake test as described above.