125i cck 8

The WT or mutant CCK_ARs were transiently transfected into HEK 293T/17 cells (purchased from the Cell Bank at the Chinese Academy of Sciences), which were cultured in a poly-D-lysine-coated 96-well plate. After 24 h, the cells were washed twice and incubated with blocking buffer (Dulbecco’s modified Eagle medium (DMEM) supplemented with 33 mM HEPES, and 0.1% (wt/vol) bovine serum albumin (BSA), pH 7.4) for 2 h at 37 °C. After three washes with ice-cold phosphate-buffered saline (PBS), the cells were treated by a constant concentration of ¹²⁵I-CCK-8 (40 pM, PerkinElmer) plus eight different doses of CCK-8 (1 pM to 10 μM) for 3 h at r.t. Cells were washed three times with ice-cold PBS and lysed by 50 μl of lysis buffer (PBS supplemented with 20 mM Tris-HCl and 1% (vol/vol) Triton X-100, pH 7.4). Subsequently, the plates were counted for radioactivity (counts per minute) in a scintillation counter (MicroBeta^{2 (link)}
plate counter, PerkinElmer) using 150 μl of scintillation cocktail (OptiPhase SuperMix, PerkinElmer).