For time-lapse microscopy, the Flp-InTRex293 cells that carried a single copy of wild-type or various mutant CCR6 with C-terminal GFP-tag were treated with CCL20 (50 nM) in FluoroBrite medium (Gibco) while recording by timelapse microscopy for 30 minutes (Online Supplementary Figure S4). The CCR6 expression was quantified using Image J (https://theolb.readthedocs.io/en/latest/imaging-/measuring-cell-fluorescence-using-imagej.html).
For flow cytometry analysis, the Flp-InTRex293 cells with a single copy of the wild-type or mutant CCR6 expression construct were treated with CCL20 (50 nM), and an aliquot (100 mL) was taken out at the indicated times for measuring surface CCR6.10 (link),19 (link)
For flow cytometry analysis, the Flp-InTRex293 cells with a single copy of the wild-type or mutant CCR6 expression construct were treated with CCL20 (50 nM), and an aliquot (100 mL) was taken out at the indicated times for measuring surface CCR6.10 (link),19 (link)
