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. 2023 May 30;18(5):e0285597. doi: 10.1371/journal.pone.0285597

Fig 5. AM scavenging in HEK293 cells requires co-expression of RAMP2/3 and CALCRL but is independent of ACKR3.

Fig 5

(A) ACKR3 is expressed in HEK293-ACKR3 but not in HEK293-CTRL cells. Antibody staining with anti-human ACKR3 (clone 11G8). (B-E) HEK293-ACKR3 or HEK293-CTRL cells were incubated with CXCL11/12-AF647 or AM-AF568 at either 4°C or 37°C and uptake activity was quantified by flow cytometry. While (B, C) CXCL11/12-AF647 was avidly scavenged by HEK293-ACKR3 cells, (D, E) no evidence of uptake was observed for AM-AF568 in HEK293-ACKR3 cells. (A,B,D) Representative Histograms. (C,E) Pooled measurements of 3–4 independent experiments are shown as mean ±SD. Statistics: Paired Student’s t-test. (F, G) HEK293-ACKR3 or HEK293-CTRL cells were transiently transfected with CALCRL, RAMP2 or RAMP3 encoding plasmids and combinations thereof and AM-AF568 uptake was investigated by flow cytometry. AM-AF568 internalization was exclusively dependent on co-expression of CALCRL with (F) RAMP2 or (G) RAMP3, regardless of ACKR3 expression. (F,G) Pooled measurements from 4 independent experiments are shown as mean ±SD. RM two-way ANOVA , Šídáks multiple comparison test. A p-value of p≥0.05 was considered not significant (ns).