Fig. 3. A 1148 amino acid, C-terminal fragment of CA125, CA125-C-TERM, retains the ability of CA125 to bind specifically to galectin-1. CHO and HeLa cells were induced to express CA125-C-TERM by retroviral transduction. For comparison, CHO and HeLa cells were included that were treated with retroviral control particles. A detergent lysate of the cells was prepared and incubated with either GST—galectin-1-, GST—galectin-3 or GST beads. Following extensive washing, the beads were treated with lactose. Eluted proteins were separated on 10% Novex NuPage Bis-Tris gels followed by transfer to a blotting membrane. CA125-C-TERM was then detected by OC125 staining using electrochemiluminescence (A). For comparison, the pattern of CA125-derived fragments isolated from HeLa cells is shown in the leftmost lane (control). (B) The intensity of CA125-C-TERM-derived bands was quantified using Bio-Rad® QuantityOne® software. (C) A crosslinking experiment is shown employing disuccinimidyl glutarate (DSG). CA125-C-TERM-expressing CHO cells were lysed with detergent followed by incubation of the cell-free supernatant with GST—galectin-1 beads. After extensive washing, DSG was added at a final concentration of 0.5 mM. Crosslinking products were eluted with SDS sample buffer and analysed by SDS-PAGE and western blotting employing affinity-purified anti-galectin-1 and monoclonal anti-CA125 antibodies. The square bracket indicates crosslinking products with an apparent molecular mass of about 160-180 kDa positive for galectin-1 and CA125. In the range of 120-130 kDa, other galectin-1-containing crosslinking products are observed.

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