Anti-Human CCR5 Recombinant Antibody (MC-1) (CAT#: TAB-1540CL)

Figure 1 Immunostaining of CCR5-CCR2b chimeras and CCR5 point mutants with anti-CCR5 mAbs.

CCR5-CCR2b chimeras are coded according to the origin of the N-terminal domain (first digit), and of the three extracellular loops (last three digits): 5555 and 2222 represent CCR5 and CCR2b, respectively; 2555 represents a chimeric receptor containing the amino-terminal domain of CCR2b and the three extracellular loops of CCR5. CCR5 point mutants are designated by the nature of their amino acid substitution. CHO-K1 cells stably expressing these constructs were stained with saturating concentrations of the different anti-CCR5 mAbs and phycoerythrinconjugated anti-mouse IgG, and analyzed by FACS. Fluorescence histograms are shown together with the background fluorescenceobtained for cells expressing CCR2b.

Blanpain, C., Vanderwinden, J. M., Cihak, J., Wittamer, V., Le Poul, E., Issafras, H.,... & Parmentier, M. (2002). Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies. Molecular biology of the cell, 13(2), 723-737.

Figure 2 MC-1 mAb induces CCR5 endocytosis in CHO cells.

CHO cells expressing CCR5 were incubated with MC-1 (■]), MC-4 (□) (A), or RANTES (■) (B) for 30 min at 37°C. Cell surface CCR5 was detected by flow cytometry with a saturating concentration of the same mAbs or MC-4 for RANTES-induced CCR5 down-modulation. Results were normalized for the fluorescence of unstimulated cells (100%) and for the background fluorescence (0%). All experiments were repeated at least twice with similar results.

Blanpain, C., Vanderwinden, J. M., Cihak, J., Wittamer, V., Le Poul, E., Issafras, H.,... & Parmentier, M. (2002). Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies. Molecular biology of the cell, 13(2), 723-737.

Figure 3 MC-1 induces CCR5 endocytosis by an arrestin and clathrin-independent, caveolae-dependent pathway.

(A-L). CHO-K1 cells expressing CCR5-GFP were incubated for 18 h with 100 ng/ml pertussis toxin 100 (A-D) or for 1 h with 0.45 M sucrose (E-H), or or 2.5 μg/ml filipin (I-L) for 1 h at 37°C. Cells were then stimulated with 100 nM RANTES (A, B, E, F, I, and J) or 10 μg/ml MC-1 (C, D, G, H, K, and L), and the dynamics of CCR5-GFP was recorded by confocal microscopy. Responses are shown before (A, C, E, G, I, and K) and 30 min after (B, D, F, H, J, and L) ligand addition. (M-P). CHO-K1 cells stably expressing CCR5 were transfected with β-arrestin-GFP; 24 h after transfection, subcellular redistribution of β-arrestin-GFP was analyzed by confocal microscopy. The cells were exposed to 100 nM RANTES(M and N) or 10 μg/ml MC-1 (O and P). Images were acquired every 15 s for 10 min. Responses are shown before (M and O), and 2 min (N) or 10 min (P) after ligand addition. Videos of the dynamics of β-arrestin-GFP trafficking in response to RANTES stimulation, as described in this figure, are available in the online version of this article. All experiments were repeated at least twice with similar results. Bar, 20 μm.

Blanpain, C., Vanderwinden, J. M., Cihak, J., Wittamer, V., Le Poul, E., Issafras, H.,... & Parmentier, M. (2002). Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies. Molecular biology of the cell, 13(2), 723-737.

Figure 4 293T cells expressing CCR5-luc and CCR5-YFP were incubated with coelenterazine, and the luminescence and the fluorescence signals were quantified before and after the addition of the indicated mAb.

The BRET ratio was defined as [(emission at 485 nm ± 20)/(emission at 530 nm ± 25)]-[(emission at 485 nm ± 20)/(emission at 530 nm ± 25)] for CCR5-luc expressed alone in the same experiments. Variation in BRET signal compared with baseline (addition of buffer alone) after 10 min of incubation with antibodies is displayed. All experiments were repeated at least twice with similar results. All points were performed in triplicates (error bars: SEM).

Blanpain, C., Vanderwinden, J. M., Cihak, J., Wittamer, V., Le Poul, E., Issafras, H.,... & Parmentier, M. (2002). Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies. Molecular biology of the cell, 13(2), 723-737.