Fig. 5
IH-CCL5 induced CCR5 downmodulation and internalisation. CCR5 downmodulation was assessed through the loss of anti-CCR5 MC5 binding at 37 oC. (a,b) Flow cytometry histogram overlays for MC5 (detected with an anti-mouse A647; APC channel) in different conditions (Medium – Solid grey line with grey fill, 100 nM CCL5 – Blue solid line, 100 nM CCL5 + 800 nM TAK-779 – Red solid line and isotype control – Grey dotted line). (c) Change in specific MFI following treatment with the indicated recombinant CCL5 alone (black bars) or with TAK-779 pre-treatment (Grey bars) compared to untreated cells (n = 3). ** P ≤ 0.0021 and **** P ≤ 0.0001 One-way ANOVA with Bonferroni test. (d) Comparison of CCR5 downmodulation induced by IH-CCL5 and commercial CCL5 for CHO-CCR5 cells treated with 100 nM chemokine 1 h at 37oC (n = 3). T-test showed no significant difference (ns). (e) IH-CCL5 induced CCR5 internalisation using immunofluorescence microscopy. CHO-CCR5 cells were prelabelled with MC5 (5 µg/mL) followed by 100 nM chemokine treatment 0.5 h 37oC. Secondary anti-mouse A568 4 µg/mL (red) and cells mounted in mowiol containing DAPI (blue). Confocal microscopy images (scale bar at 20 μm) analysed on ImageJ.
