Anti p22phox

After isolation from whole blood, 100 × 10⁶ neutrophils were resuspended in 5 ml of HBSS^+/+ and incubated with or without T2AA (25 or 50 µM) at 37°C for 1 h. Neutrophils were then stimulated with PMA (200 ng/ml) for 8 min while the mixture was gently shaken. Ice-cold buffer was added, and cells were pelleted by centrifugation at 400 g for 8 min at 4°C. The pellets were resuspended in relaxation buffer containing 1 mM ATP, 1 mM EGTA, 0.5 mM PMSF, 25 nM Calyculin, and 5 µg/ml of aprotinin, leupeptin, and pepstatin. The cells were then disrupted by sonication (twice for 10 s each time) at 4°C and centrifuged at 400 g for 8 min. The postnuclear supernatant was loaded onto a discontinuous sucrose gradient (35 and 15% sucrose) diluted in relaxation buffer and centrifuged for 45 min at 150,000 g. The cytosolic and membrane fractions (in the upper and the intermediate layers, respectively) were collected. Proteins were analyzed by Western blot analysis using anti-p47phox (BD Transduction Laboratories), anti-p22phox (Santa Cruz), anti-phospho-p47phox, and anti-PCNA (clone PC10; Santa Cruz) mouse mAbs. Expression of p22phox and total p47phox served as a loading control in membrane fraction and cytosolic fraction, respectively.