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. 2022 Feb 22;7(4):e151892. doi: 10.1172/jci.insight.151892

Figure 2. Human monocyte-derived macrophages express DAT and NET that are localized to the plasma membrane.

Figure 2

(A) Representative immunoblots for NET and DAT from macrophage intracellular and membrane fractions separated via biotinylation assay (splice sites indicated by solid lines). (B) Representative 40× total internal reflective fluorescence microscopy (TIRF-M) images of cultured macrophages labeled with CTxB-555 and either NET or DAT antibodies. CTxB-555 punctae appeared on the basal membrane, along with scattered punctae of NET (top) and DAT (bottom). Some punctae of CTxB colocalized with NET or DAT (arrowheads, n = 4 experiments). (C and D) Representative confocal images (C, n = 4 experiments) or stimulated emission depletion (STED) images (D, n = 2 experiments) on cultured human macrophages labeled with CTxB-555 and for DAT with some of the DAT signal colocalizing with the CTxB-555 signal at or near the membrane. (E) Schematic of the experimental design for JHC1-064 binding assay to identify NET-specific and DAT-specific binding. (F and G) Live-cell confocal (60×) and TIRF-M (40×) images of JHC1-064 binding to macrophages in conditions shown in E. (H) Quantifying the JHC1-064 signal from F shows that blocking NET or DAT decreased JHC1-064 binding. Blocking both transporters further decreased the JHC1-064 signal on macrophages (2-way ANOVA with Tukey’s test for multiple comparisons, P < 0.05). (I and J) The magnitude (I) and rate (J) of JHC1-064 binding to human macrophages were decreased by the addition of Nom or DMI. (I) Control versus DAT-specific (P < 0.0001), control versus NET-specific (P < 0.0001). (J) Control versus DAT-specific (P = 0.0004), control versus NET-specific (P < 0.0001). Blocking all 3 transporters further decreased the magnitude (I, P < 0.0001) and rate (J, P < 0.0001) of JHC1-064 binding. Images and data in F–J are from n = 114–157 cells/group from 3 independent experiments. Statistical analysis in I and J performed by Kruskal-Wallis test with Dunn’s test for multiple comparisons.