560141.fig.001a
(a)
560141.fig.001b
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560141.fig.001c
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560141.fig.001d
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560141.fig.001e
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Figure 1: Localization and activity of native SLPI in peripheral blood neutrophils. (a) Coomassie blue stained gel of isolated neutrophils subjected to subcellular fractionation yielding a cytosol fraction (lane 1), membrane fraction (lane 2), and secondary and primary granule fractions, lane 3 and 4 respectively. Western blotting employed polyclonal rabbit (Rb) or goat (Gt) antibody to SLPI, which localized SLPI to both the cytosolic and secondary granule fractions. As controls, MPO and lactoferrin were detected using rabbit polyclonal antibodies as markers for primary and secondary granules, respectively. (b) Coomassie blue stained gel of isolated CF and COPD neutrophils subjected to subcellular fractionation yielding a cytosol fraction (lane 1), membrane fraction (lane 2), and pooled primary and secondary granule fractions (lane 3). Western blotting employed polyclonal Rb antibody confirming cytosolic localization of SLPI in patient samples. (c) The NE inhibitory activity of neutrophil cytosol was assessed before and after immunoprecipitation (IP) of SLPI using Gt polyclonal anti-SLPI antibody (Synergen). The kinetics of inhibition is illustrated in (c), and data of the final time point (240 sec) is plotted in (d). Control experiments included isotype control Gt IgG. * between NE control. (e) Addition of 480 nM rhSLPI to cells (2 × 105/mL) increased the concentration of cytosolic SLPI by approximately 50% above the untreated cells (Con), as determined by ELISA. Experiments were repeated at 4°C or in the presence of NaN3 (15 mM) and NaF (10 mM). * between untreated cells (con). Results illustrated in (a) and (b) are representative gels and blots of 3 separate experiments. Results illustrated in panel (c)–(e) were performed in triplicate and each bar is the mean ± S.E. (NS, no significant difference, * calculated by Student’s -test).