Fig. 3.

Galectin-3 is associated with CD13 in U-937 cells. (a) Binding of the anti-CD13 mAb 452 to U-937 cells pre-incubated or not with the anti-galectin-3 antibody for 1 h at 37 °C. (b) HA was induced with the anti-CD13 mAb for 2 h using 1x10⁷ cells/ml. Antibody treated (HA) or untreated, non-aggregated cells (NA) were lysed and CD13 (IP CD13) or galectin-3 (IP gal-3) was immunoprecipitated as described in Materials and methods. A Western-blot was performed using anti-CD13 (WB CD13) and subsequently, the same membrane was re-probed for galectin-3 (WB gal-3). Immunoprecipitation of CD13 resulted in co-immunoprecipitation of galectin-3 but in lower levels in aggregated (lane 3) than non-aggregated cells (lane 1). Immunoprecipitation of galectin-3 also resulted in co-immunoprecipitation of CD13 (lane 2). Anti galectin-3 immunoprecipitates from HA cells contain CD13 because of the anti-CD13 antibody used to induce HA (lane 4). (c) Membrane expression of CD13 in NA and in HA cells that had been pre-incubated in the presence (HA+anti-gal-3) or absence (HA) of anti-galectin-3 antibodies. (d) Membrane expression of galectin-3 was determined by flow cytometry in HA and NA cells. (e) Cells were incubated with anti-CD13 mAb at 4 °C. After washing, they were co-incubated at 37 °C with calcein-labeled cells which had not been pre-incubated with anti-CD13 mAb. Images were taken after 4 h. (f) Same experiment as in (e) but the calcein-labeled population was pre-incubated with anti-galectin-3 antibodies before HA.