Fig 2: CXCR4 blocking assays.a. Total cell lysate from MNB-OZ, NB 19 and SK-N-SH cells were electrophoresed and blotted on to PVDF membrane, and protein detection was carried out with anti-47 kDa neutralizing antibody. Shown in figure is the representative result from 5 experiments. b. 80% confluent SK-N-SH cells were harvested and incubated in either RPMI, RPMI + IgG control, RPMI + AMD 3100 or RPMI + anti-47 kDa neutralizing antibody. The supernatants were harvested after 24 hours, centrifuged and subjected to gelatin zymography. The gels were analyzed using imageJ and the relative MMP-9 secretion was calculated from 3 experiments (* p < 0.05). c. Total cell lysates from SK-N-SH cells either serum-starved (control), treated with IgG control (negative control), RPMI + AMD 3100 or RPMI + anti-47 kDa neutralizing antibody was subjected to western blot analysis. Protein detection was carried out with anti-integrin a3 or anti-integrin ß1 antibodies. Anti-actin antibody was used for loading control. Shown in figure is the representative result from 3 experiments. d. Transwell invasion studies were carried out with cell lines SK-N-SH, MNB-OZ and NB 19. Cells were serum-starved (control), treated with 10 µM AMD 3100 and treated with anti-47kDa CXCR4 neutralizing antibody. Plotted is the mean number of cells migrating across the mesh. (* p<0.05) e. Invasion index of cell lines SK-N-SH, MNB-OZ and NB 19, serum-starved (control) and treated with AMD 3100 and anti-47 kDa CXCR4 neutralizing antibody, were analyzed using transwell invasion assays. Plotted is the normalized invasion index values obtained from 5 experiments (* p < 0.05).

Fig 3: Effect of AMD3100 on binding and functional properties of cells expressing CXCR4 and ChemR23.[A, B] Competition binding assays were performed on cells expressing CXCR4 (•) and ChemR23 (?) only, or cells co-expressing CXCR4 and ChemR23 (?). Cells were incubated with 0.1 nM 125I-CXCL12 (A) or 125I-[145–157]-chemerin (B), as tracers and increasing concentrations of unlabelled AMD3100 as competitor. After one hour incubation, unbound tracers were separated by filtration and filters washed twice before counting. The data were normalized for nonspecific binding (0%) in the presence of 300 nM of unlabelled CXCL12 or chemerin respectively, and specific binding in the absence of competitor (100%). All points were run in duplicate (error bars indicate S.E.M.). The displayed data are representative of two independent experiments. The functional responses of CHO-K1 cells expressing CXCR4 (C), ChemR23 (E) or both receptors (D, F) were measured by using the aequorin-based calcium mobilization assay. [C, D] Cells preloaded with coelenterazine were stimulated with increasing concentrations of CXCL12 (?) or CXCL12 + AMD3100 (?). [E, F] Cells preloaded with coelenterazine were stimulated with increasing concentration of chemerin (•) or chemerin + AMD3100 (?). The results were normalized for baseline activity (0%) and the maximal response obtained with 25 µM ATP (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of three independent experiments.

Fig 4: Aequorin-based functional assay in cells co-expressing ChemR23 with CCR7 or CXCR4.The functional responses of CHO-K1 cells expressing ChemR23 (?), CXCR4 (•), CCR7 (?), ChemR23 + CXCR4 (?) or ChemR23 + CCR7 (?) were measured using the aequorin-based calcium mobilization assay. Cells pre-loaded with coelenterazine H were stimulated with increasing concentrations of chemerin (A, C), CXCL12 (B) or CCL19 (D) and luminescence was recorded for 30 s. The results were normalized for baseline activity (0%) and the maximal response obtained with 25 µM ATP (100%). All points were run in triplicates (error bars indicate S.E.M.). The displayed data are representative of three independent experiments.

Fig 5: Structural heterogeneity of CXCR4 in neuroblastoma cell lines.a. Western blot detection of CXCR4 in 20 neuroblastoma cell lysates. 1 X 107 cells were washed twice in PBS and solubilized in 1 ml of lysis buffer. The lysates were centrifuged at 14,000 rpm at 4°C for 30 minutes. Equal amounts of total proteins, as determined by BCA assay, were loaded on to a 12.5% polyacrylamide gel and electrophoresed. The proteins were then blotted on to a PVDF membrane and CXCR4 was detected using an anti-CXCR4 polyclonal antibody and a HRP conjugated secondary antibody. Shown in figure is a representative result from 5 experiments. b. The bands from western blot studies were quantified using imageJ. Plotted is the mean relative expression of CXCR4 isoforms in the whole cell lyaste from 5 experiments. c. CXCR4 was immune precipitated using the Pierce Crosslink Immunoprecipitation kit with anti-CXCR4 antibody. The precipitate was electrophoresed and blotted on to PVDF membrane. Protein detection was carried out using a second anti-CXCR4 antibody. Shown in figure is a representative result from 5 experiments. d. The bands from western blot studies were quantified using imageJ. Plotted is the mean relative expression of CXCR4 isoforms in the CXCR4 immune precipitate from 5 experiments.