Figure 7.

Differences in ACKR3 recycling after stimulation with CXCL12 or CXCL11 are abolished in the R142A mutant. A, direct labeling of surface receptor with anti-ACKR3 antibody 358426 coupled to APC. Transfected HEK293 cells were preincubated for 2 h with 50 μm cycloheximide (0). They were then stimulated for 15 min with vehicle (control) or 200 nm chemokines (stim), before digestion of remaining surface receptor and chemokine with proteinase K on ice (pK). The cells were then washed and shifted to 37 °C for 15, 45, or 180 min. Note the absence of rapid reappearance of ACKR3 after CXCL11 stimulation at 15 and 45 min, but not 180 min (ANOVA with Tukey's post hoc test at the indicated time point: *, p < 0.05 between CXCL11 and CXCL12). B and C, recycling rates of R142A (bold color) compared with wildtype ACKR3 (pale color), after stimulation with CXCL12 (B, red symbols) or CXCL11 (C, blue symbols). The time point 0 in B and C corresponds to the point pK in A. **, p < 0.01, t test between ACKR3 and R142A. All data are from three independent experiments. Error bars, S.D.