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. 2011 Oct 18;6(10):e26560. doi: 10.1371/journal.pone.0026560

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Carlsson et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A) The concentrations of IgM in the galectin-1 bound fractions from cancer (n = 25) and healthy sera (n = 25) as determined by direct enzyme-linked immunosorbent assay (ELISA). The average for each group is marked by horizontal lines, but the slight difference was statistically not significant (p = 0.41). B) Initial serum levels of IgM (Y-axis) compared to % of galectin-1 bound IgM (X-axis). The averages and ranges of each parameter are shown by crossed lines, unbroken for cancer sera and dashed for healthy sera. The difference between the % galectin-1 bound IgM was significant (p<0.0001) but the difference between total IgM was not (p = 0.24). The three samples with no galectin-1 bound IgM (seen as dots on Y axis) were excluded from the calculation of averages, ranges and significances shown; including them made only a minor change in averages and no change in significance of differences. C) The concentrations of haptoglobin in the galectin-1 bound fractions from cancer (n = 25) and healthy sera (n = 25) as determined by direct ELISA. The average for each group is marked by horizontal lines, and the difference was statistically significant (p<0.0001). Error bars represent SEM from three independent duplicate measurements for each data point. D) Initial serum levels of haptoglobin (Y-axis) compared to % of galectin-1 bound haptoglobin (X-axis). The averages and ranges of each parameter are shown by crossed lines, unbroken for cancer sera and dashed for healthy sera. The difference between the % galectin-1 bound haptoglobin was significant (p<0.0001) and the difference between total haptoglobin was also significant (p = 0.0058). E) Chromatograms of 2 mg purified pooled haptoglobin separated by affinity chromatography on columns (1 ml) with immobilized galectin-1 C3S. The protein concentration of each fraction (0.2 or 1 ml) is given on Y-axis. Elution with lactose (150 mM) started after washing with 32 ml PBS (arrow heads). Non-binding haptoglobin was chromatographed a second time but only traces (∼1%) of additional haptoglobin was now found in the bound fraction. The chromatogram from the second run has been moved for clarity by +0.05 on Y-axis and by +5 on X-axis.