Figure 5.

Identification of galectin-3 as an NG2-binding protein. (A) Identification of NG2-interacting proteins. Extracts from MAEC were incubated with either NG2 beads (lane 3) or CL-4B Sepharose beads as a control (lane 2). Bound proteins were visualized on 4–12% Tris-glycine gels by silver staining. NG2 beads alone were also loaded as another control (lane 1). MAEC proteins bound specifically to NG2 are labeled with arrowheads. The 30-kDa protein band (arrow) was analyzed by MALDI-TOF mass spectrometry. (B) Amino acid sequence of galectin-3. Shaded sequences indicate tryptic peptides derived from the 30-kDa band, as determined by mass spectrometry. (C) MAEC proteins bound to NG2 beads (lane 2) or CL-4B Sepharose beads (lane 1) were analyzed by immunoblotting with antigalectin-3 antibody. A crude lysate of MAEC was also loaded (lane 3). (D) NG2/EC⁺ or NG2/EC^- were incubated with glutathione-agarose beads carrying either GST-galectin-3 fusion protein (lane 3 and 4) or GST (lane 2) as a control. Bound proteins were eluted from GST-galectin-3 fusion protein beads with 100 mM lactose (lane 3), and the stripped beads were boiled in SDS-PAGE loading buffer (lane 4). Samples were separated by SDS-PAGE, and immunoblotted using anti-NG2 antibody. (lane 1) NG2/EC was loaded as a positive control. NG2/EC⁺ migrates as a broad smear because of the presence of the chondroitin sulfate chain. NG2/EC^- appears as three distinct bands because of proteolytic degradation during its purification. (E) GST-galectin-3 fusion protein was incubated with either NG2 beads (lane 3 and 4) or control CL-4B beads (lane 2). After washing, bound proteins were eluted from NG2 beads with lactose (lane 4). Stripped beads were also analyzed (lane 3). Samples were treated as described above and immunoblotted using antigalectin-3 antibody. (lane 1) GST-galectin-3 fusion protein was loaded as a positive control.