Fig. 1. Identification of CA125 as a counter receptor of galectin-1. (A) Affinity
purification of galectin-1-interacting proteins. Both soluble (lanes 1, 2, 5,
6) and membrane (lanes 3, 4, 7, 8) fractions of HeLa cells were incubated with
either GST—galectin-1 beads (lanes 2, 4, 6, 8) or GST beads as a control
(lanes 1, 3, 5, 7). Bound proteins were eluted sequentially with lactose
(lanes 1-4) and glutathione (lanes 5-8), followed by separation on Novex
NuPage 10% Bis-Tris gels. Protein bands were visualized using SilverQuest. (B)
Immunoblot analysis of the proteins eluted from the GST—galectin-1 and
GST matrices, respectively. The fractions were loaded in the same order as
shown in panel A. The anti-CA125 antibody OC125 was used as primary antibody
followed by detection by electrochemiluminescence. (C) Immunoblot analysis as
shown in panel B employing an anti-CA125-C-TERM1-356 antiserum for
the detection of CA125-derived fragments. (D) Amino acid sequence of
CA125-C-TERM. Boxed sequences indicate tryptic peptides derived from band 1
(panel A, lane 2) as identified by mass spectrometry.