LPS-Induced Galectin-3 Oligomerization Results in Enhancement of Neutrophil Activation (original) (raw)
Figure 5
LPS induces oligomerization of Gal 3.
A: A 96-well microplate was coated with 1 µg of recombinant Gal 3 per well. After blocking with gelatin 3%, 0.2 µM biotin-labeled Gal 3 ([b]Gal 3) and the indicated concentrations of unlabeled Gal 3 were added to each well in the presence (solid squares) or absence (open squares) of 5 µg/mL E. coli LPS. The signal was generated by neutravidin-HRP using OPD as a substrate. B and C: PMNs (2×106/mL) were incubated for 30 min at 4°C with increasing concentrations of truncated Galectin-3 (Gal 3C), lacking the N-terminal part (B), or increasing concentration of full-length Galectin-3 (Gal 3) (C). After washing, a specific polyclonal goat anti-Gal 3C (B) or goat anti-Gal 3 (C) and an FITC-labeled secondary anti-goat IgG antibody were used to detect Gal 3C on neutrophils by flow cytometry (n = 2). D: PMNs (10×106/mL) were treated for 20 min at 37°C with FITC-LPS (12.5 µg/mL) preincubated with Gal 3 (8 µM) or with Gal 3C (8 µM). The binding of FITC-LPS was evaluated by flow cytometry as in figure 4. E: PMNs (2×106/mL) were treated for 45 min at 37°C with the indicated concentrations of full-length Gal 3 (Gal 3) or Gal 3C, preincubated or not with E. coli LPS (1 µg/mL), and analyzed for CD11b expression by flow cytometry. ***p<0.001.