Subcellular localization of galectin-3–binding proteins in neutrophils. The organelles were obtained by fractionation of disintegrated peripheral blood neutrophils on a three-step Percoll gradient (α, β1, and β2)30 and a flotation gradient (γ1 and γ2),29 respectively (Fig 7). A Coomassie-stained SDS-polyacrylamide gel (5% to 20%; a) with separated proteins from the neutrophil plasma membrane (γ2), secretory vesicles (γ1), gelatinase granules (β2), specific granules (β1), and azurophil granules (α), respectively, corresponding to the fractionated content of 5 × 10⁶cells, is shown together with corresponding Western blots (b and c). The blots were incubated with galectin-3 (8 μg/mL) in the absence (b) or presence (c) of lactose (100 mmol/L), followed by incubation with antibodies directed against galectin-3 (anti–Mac-2 antibodies; culture supernatant from the hybridoma M3/38; 1/25) and finally with HRP-labeled rabbit anti-rat Ig antibodies (DAKO P0450; 1/1,000). The blots were developed with peroxidase substrate (VIP Kit; Vector). Molecular sizes are given in kilodaltons.