Myrcludex B pulse-chase experiment

U2OS cells of 80% confluency in 96-well format were incubated with 200 nM Myrcludex B-FITC for 30 min at 37°C. Subsequently, cells were washed 3 times with PBS and complete medium was added for the indicated chase period. After incubation, cells were washed 3 times with PBS and fluorescence intensity was measured using a Clariostar microplate reader (BMG Labtech GmbH, Offenburg) at λex / λem = 483-14 nm/ 530-30 nm. The option orbital averaging was set at a diameter of 3 mm. Background fluorescence, the Myrcludex B-FITC signal obtained in parental U2OS cells, was subtracted from the HA-hNTCP expressing U2OS cells and results were plotted as percentage of 0 h incubation.