Analysis of PR3 subcellular localization by neutrophil fractionation. (A) Measurement of PR3, HNE, and MPO in fractionated neutrophils. Resting neutrophils from an individual having an 80% mPR3⁺ subset were fractionated into the plasma membrane-enriched fraction that contains secretory vesicles, the specific granules, the azurophil granules, and the cytosol. Double sandwich ELISA were used to specifically quantify PR3, HNE, and MPO in an aliquot of each fraction equivalent to 50 × 10⁶neutrophils. The histogram depicts the percentage of each protein in the different fractions. Data are the mean ± SEM of 4 determinations obtained in a representative fractionation experiment. (B) Western blot analysis of the membrane-enriched fraction as compared with azurophil granules. The neutrophil membrane-enriched fraction (100 × 10⁶ neutrophils) washed with high salt concentration buffer (50 mmol/L Tris, 3 mol/L NaCl) before analysis was compared with purified azurophil granules (10 × 10⁶ neutrophils).