CXCR4 blocking assays.

<p>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 α3 or anti-integrin β1 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).