Fig. 2. CA125 displays differential binding efficiency towards galectin-1 when compared with galectin-3. (A) Soluble (lanes 1-4) and membrane fractions (lanes 5-8) were prepared from HeLa cells followed by the incubation with either GST—galectin-1 (lanes 1, 3, 5, 7) or GST—galectin-3 (lanes 2, 4, 6, 8) beads. The amounts of GST—galectin-1 and GST—galectin-3 fusion proteins, respectively, used for affinity purification of CA125-derived fragments were shown to be comparable by western blotting employing affinity-purified anti-GST antibodies (lanes 9 and 10). Following extensive washing, bound proteins were eluted sequentially with lactose (lanes 1, 2, 5, 6) and glutathione (lanes 3, 4, 7, 8). Eluted proteins were separated on 10% Novex NuPage Bis-Tris gels and transferred to a blotting membrane. Electrochemiluminescence detection of CA125-derived fragments was performed employing the mAb OC125. (B) Quantitative analysis of CA125-derived fragments in the fractions shown in panel A employing Bio-Rad® QuantityOne® Software. (C) Total protein pattern of lactose-eluted proteins derived from the galectin-1 matrix (lane 1) and the galectin-3 matrix (lane 2). Eluted proteins were separated on NuPage Bis-Tris gels followed by silver staining according to standard procedures. Labels indicate examples for proteins that preferentially bind to galectin-1 (•), galectin-3 () or proteins that equally bind galectin-1 and galectin-3 ().