Drugs were dissolved in DMSO to a concentration of 1 mM and further diluted in HBSS supplemented with 10 mM HEPES. ciBECs were cultured onto cell-culture inserts as described above. We refreshed 50 μL of pre-warmed HBSS supplemented with 10 mM HEPES and 0.1% BSA by equilibration to 37°C for 15 min. At the time point 0 min, 50 μL of 4 μM drug was added to the upper chamber of the inserts, which were then transferred to 24-well plates containing 1 mL of pre-warmed HBSS supplemented with 10 mM HEPES. Samples were collected from the lower chamber at 10, 20, 30, and 40 min, 15, 30, 45, and 60 min, or 30, 60, 90, and 120 min. Samples were desalted with reversed-phase StageTips (Rappsilber et al., 2007) and suspended in loading buffer (0.5% trifluoroacetic acid and 4% acetonitrile) for subsequent nanoLC-MS/MS analysis.
