Figure 2.

Expression of hC4.4A in cancer cell lines. (A) C4.4A expression was evaluated in colorectal cancer lines, which were cultured in heat-inactivated (grey area) or fresh ABO serum (black area). Overlays with the negative control (normal rabbit IgG/anti-rabbit APC) are presented. (B) Biotinylated lysates of the indicated cell lines were immunoprecipitated using anti-hC4.4A-C. Immunoprecipitates were boiled, separated by SDS–PAGE and blotted with streptavidin-HRP. Depending on the cell line, several bands were recovered (indicated by arrows), which represent different C4.4A isoforms and/or associated proteins (see Results). (C) Biotinylated lysates of Colo357 and MCF-7 cells were immunoprecipitated using anti-hC4.4A-C, and immunoprecipitates were boiled in the presence of 2-ME before SDS–PAGE and blotting with streptavidin-HRP. The high molecular weight band of about 200 kDa was not recovered after 2-ME treatment. (D) Where indicated, MCF-7 cells were treated with tunicamycin (Tun). Lysates were separated by SDS–PAGE and blotted with anti-hC4.4A-N. The antibody recognizes only lysates from Tun-treated cells. (E) Lysates of MCF-7 cells were biotinylated and immunoprecipitated with the anti-hC4.4A-C antibody. Where indicated, immunoprecipitates were treated with N-glycosidase F (N-glycosid.) or with O-glycosidase (O-glycosid.) plus neuraminidase (NA) before SDS–PAGE separation and WB analysis with streptavidin-HRP. O-glycosidase plus NA treatment resulted in a size reduction of C4.4A from 75 to 35 kDa–40 kDa. (F) Cell lysates of the indicated lines were incubated with GST-galectin-3 or GST. Bound proteins were eluted with 100 mM lactose. Eluted proteins were treated with N-glycosidase F before SDS–PAGE and blotting with anti-hC4.4A-N. C4.4A with a molecular weight of about 55 kDa was recovered in the precipitate of 5 out of 7 lines, but only after incubation with GST-galectin-3 and not after incubation with GST.