560141.fig.008a
(a)
560141.fig.008b
(b)
560141.fig.008c
(c)
560141.fig.008d
(d)
560141.fig.008e
(e)
Figure 8: SLPI is secreted from the cell upon activation. (a) Coomassie Blue stained gel (top panel) and immunoblots (bottom 2 panels) employing anti-SLPI antibody (Gt, Synergen) for its respective distribution (intracellular or extracellular) in control unstimulated cells (Con) and after IL-8 (1.2 nM) and fMLP (1 μM) or PMA (1.6 μM) activation for 10 min. (b) Donor neutrophils (5 × 106/mL) were stimulated with PMA (1.6 μM) for 0, 0.5, 1, or 10 min before sonication and preparation of membrane (top panels) and extracellular released protein fractions (bottom panels). SLPI and p translocation was analysed by Western blotting employing polyclonal rabbit antibodies. (c) The distribution of SLPI in resting unstimulated cells (Con) and after PMA or fMLP (1 μM) and IL-8 (1.2 nM) activation for 10 min was detected using an FITC labeled rabbit polyclonal anti-SLPI antibody (green fluorescence). The distribution of SLPI was predominantly cytosolic in unstimulated cells, and after stimulation condensed around the margin of the cell (indicated by white arrow). DAPI stained nuclei are represented in blue ( 40 magnification, 10 zoom). (d) Coomassie blue stained gel (top panel) and immunoblot (bottom panel) employing rabbit anti-SLPI antibody for detection of released SLPI in the extracellular supernatants of unstimulated (Un) or fMLP/IL-8 stimulated (Stim) healthy control (Con), COPD or CF cells (5 × 106/mL). (e) Neutrophils isolated from healthy donors were incubated at 37°C and were treated with fMLP/IL-8. Cell free supernatants were collected at 0, 10, 20, 30, 40 or 50 sec and Western blotted for cell secreted SLPI (top panel) and intracellular cytosolic Ca2+ was analysed using the Invitrogen Fluo-4 Calcium Assay kit. Results of three separate experiments were expressed as relative densitometry units (bar graph), and each bar is the mean ± S.E. * is between 0 time point and statistical significance was calculated by Student’s -test.