Figure 6.

Internalization of prelabeled surface ACKR3. A, confocal microscopy of HEK293 cells co-expressing wildtype or mutant ACKR3 (bearing an N-terminal FLAG tag but no C-terminally fused YFP tag) and β-arrestin2 mCherry (red fluorescence). The top row shows surface labeling of the N-terminal FLAG tag and revelation with secondary Alexa Fluor® 488-conjugated antibody (green fluorescence) at 4 °C. The other rows show secondary labeling of permeabilized cells after 90 min of rapid temperature shift to 37 °C in the absence (control) or presence of 100 nm CXCL12 or CXCL11. B and C, quantification by flow cytometry of receptor internalization after prelabeling with uncoupled anti-FLAG antibody, followed by temperature shift and stimulation as indicated. Secondary labeling was performed at the indicated time points. Upon simultaneous curve fit, the preferred fit is a two-phase decay for ACKR3/CXCL11 (p < 0.0001), but also R142A/CXCL12 (p = 0.029) and R142A/CXCL11 (p = 0.0010), whereas a one-phase decay model is preferred in the absence of stimulation and for ACKR3/CXCL12 (ANOVA with Tukey's post hoc test at the indicated time point: *, p < 0.05 between CXCL11 and CXCL12). All data are from three independent experiments, and error bars represent S.D.