(A) Coomassie blue–stained gel of normal MM neutrophils subjected to subcellular fractionation yielding a cytosol fraction (lane 1), combined secretory vesicle/plasma membrane fraction (lane 2), and primary and secondary granule fraction (lanes 3 and 4, respectively). Western blotting employed antibodies to MPO and PR3 as markers of primary granules and antibodies against p22^phox of cytochrome b₅₅₈ as a marker of secondary granules and combined secretory vesicle/membrane fractions. AAT was localized to the secretory vesicle/membrane fraction at the interface between the 17.5/35% (w/w) sucrose. (B) By Percoll gradient fractionation, approximately 83% of AAT was localized to the plasma membrane (PM) compared with secretory vesicles (SV). As controls, HLA served as a marker of PM and HSA as a marker of SV. (C) By confocal microscopy, the distribution of AAT in non-permeabilized MM neutrophils was predominantly localized to the membrane margin of the cell. Cell nuclei are stained red. Scale bar: 10 μm. (D) Detection of AAT and flotillin-1 in the low-density lipid raft fractions (15% w/w sucrose) of human MM neutrophil lysates: untreated (–) or treated with MβCD (10 mM) (+). Immunoblots were probed with polyclonal anti-AAT and monoclonal anti–flotillin-1. (E) Coomassie blue–stained 2D SDS-PAGE gel of isolated MM neutrophil membrane lipid rafts (top panel). A Western blot probed with polyclonal anti-AAT revealed 4 associated AAT isoforms (lower panel). (F) Flow cytometry analysis of membrane-bound AAT in isolated resting MM (green) and ZZ-AATD (red) neutrophils. The level of AAT was found to be significantly higher on resting MM neutrophils. The isotype control antibody is illustrated in black (filled). Experiments in A, D, and E are each representative gels and blots of 3 separate experiments. Results in B represent experiments performed in triplicate on 3 consecutive days, and each bar is the mean ± SEM. Confocal analysis in C and FACS analysis in F are one illustrative result from 3 independent experiments.