Figure 2
Total MICL expression is reduced upon activation of human neutrophils with MSU. (A) Freshly isolated human neutrophils (20 × 106 cells/ml) were incubated with MSU (1 mg/ml) at 37°C, then the stimulation was terminated. Aliquots of the suspension were stopped at 20 min by transferring it directly into the same volume of nonreducing 2× boiling modified Laemmli sample buffer. Whole-cell lysates were probed by Western blotting for MICL (anti-MICL clone HB3; upper panel) and phosphoinositide 3-kinase (PI3K)-p85 (lower panel) as loading control. This result is representative of eight independent experiments. (B) Densitometric ratios from (A) are represented on the graph. (C) Human neutrophils (20 × 106 cells/ml) were incubated with MSU (1 mg/ml) at 37°C. Aliquots of the suspension were centrifuged, and pellets were stopped at the indicated times in nonreducing 2× boiling modified Laemmli sample buffer. The supernatants were precipitated with 2,2,2-trichloroacetic acid as described in Methods. Pellets and supernatants were probed by Western blotting for MICL (anti-MICL clone HB3). This result is representative of three independent experiments.