Figure 5.

β-arrestins contribute to, but are dispensable for CCL19 uptake. (A) Wild-type (wt) HeLa cells or β-arrestin1 and β-arrestin2 double gene targeted HeLa cells (KO) were co-transfected with ACKR4-mTq2 and β-arrestin2-YFP or empty vector, fixed, stained with phalloidin^Alexa647 and analyzed by confocal microscopy. Cell shapes are marked with a dashed line (outer-left panels). Scale bar = 25 μm or 2.5 μm for zoomed images. (B) Scheme of CCL19^Dy649P1 binding to mTq2-tagged ACKR4. (C) HeLa cells (ut, dashed line) were transfected with ACKR4-mTq2 (blue line) and incubated for 0 (t = 0) or 60 min (t = 60) at 37°C with 5 nM fluorescently labeled CCL19^Dy649P1. ACKR4-mTq2 expression and chemokine uptake (by receptor + cells) was determined by flow cytometry. One representative experiment including gating strategy is shown. (D,E) HeLa wt or HeLa KO cells over-expressing or not β-arrestin2 were transfected with ACKR4-mTq2 and incubated with 5 nM CCL19^Dy649P1 for indicated times and chemokine uptake determined by flow cytometry. Mean values and SEM (D) or SD (E) are shown (D). Cumulative chemokine uptake over time, as determined by the area under the curve (AUC) of the experiments shown in (D). Where indicated, cells were exposed to a short acidic wash to remove surface bound, but not internalized CCL19^Dy649P1. n = 4.