Supplementary Figure 1: Validation of the monoclonal antibody to mouse ACKR1 and expression of ACKR1 by BM hematopoietic cells.
From: Atypical chemokine receptor 1 on nucleated erythroid cells regulates hematopoiesis
(a to d) Comparison of immunostaining of BM cells by anti-mouse ACKR1 antibodies: new validated monoclonal (a and c, Thiriot A et al Ref. 29) and non-validated polyclonal (b and d, FAB6695P from R&D Systems used by Hur et al., Ref. 43, to report ACKR1 expression in BM macrophages). (a and b) ACKR1 immunoreactivity in nucleated erythroid cells (NECs; CD71+Ter119+), neutrophils (Neutro; CD11b+CD115–F4/80–Ly6G+), monocytes (Mono; CD11b+ CD115+F4/80–Ly6G–) and macrophages (Macro; CD11b+CD115–F4/80+Ly6G–) in BM of wild-type (WT) mice. Left, representative flow cytometry histogram plots; right, quantitative analysis. n=3. (c and d) ACKR1 immunoreactivity and negative control staining (NC) in NECs and macrophages (Macro) in BM of WT (blue) and ACKR1-deficient (KO, red) mice. Left, representative flow cytometry histogram plots; right, quantitative analysis. n=3. BM immunostaining using validated monoclonal antibody (a and c) show that ACKR1 is expressed in the BM by erythroid cells only but not by macrophages. Polyclonal non-validated antibody failed to immunoreact with NECs but at high concentration stained BM macrophages, (b) however non-specifically as (d) to the same extent in WT and KO mice. All data are Mean±SEM. One-Way ANOVA (a and b). Two-Way ANOVA (c and d). *P<0.001. (e) Relative expression of ACKR1 in hematopoietic and stromal populations as determined by microarray analysis based on the data in the BM cells transcriptome database (https://gexc.riken.jp/models). Microarray dataset of adult definitive murine erythroblasts were retrieved from ArrayExpress (https://www.ebi.ac.uk/arrayexpress; E-MTAB-1035) and implemented in Gene Expression Commons database (link to this model https://gexc.riken.jp/models/1649). Proerythroblasts (ProE), early normoblasts (BasoE), late normoblasts (PolyE), reticulocytes (Ret) and Endothelial cells (BM EC) in BM express ACKR1 whereas BM macrophages (BM Macro) do not.
