Cells were either untreated or treated with AA861 (10 μM) alone, HPU (100 nM) alone, HPU plus AA861, or IL-8 (100 nM). At least 400 cells were counted per slide. The results were expressed as mean ± S.E.M. To obtain whole cell lysates, neutrophils (5 × 106 cells/mL) were resuspended in lysis buffer (50 mM HEPES, pH 6.4, 1 mM MgCl2, 10 mM EDTA, 1% Triton X-100, 1 μg/mL DNAse, 0.5 μg/mL RNAse) containing a cocktail of protease inhibitors: 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, 1 μM leupeptin and 1 μM soybean trypsin inhibitor (all reagents from Sigma

Chem. Co., St. Louis, MO, USA). Cell lysates were denatured in sample buffer (50 mM Tris–HCl, pH 6.8, 1% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.001% bromophenol blue) and heated in a boiling water bath for 3 min. Samples http://www.selleckchem.com/products/Rapamycin.html (30 μg total protein) were resolved in 12% SDS-PAGE gels and proteins were transferred to PVDF membranes

(Hybond-P, Amersham Pharmacia Biotech). Rainbow markers (Amersham Pharmacia Biotech) were run in parallel to estimate molecular masses. Membranes were blocked with Tween-TBS (20 mM Tris–HCl, pH 7.5, 500 mM NaCl, 0.1% Tween-20) containing 1% BSA and probed with polyclonal antibodies: anti-Bcl-XL (Santa Cruz Biotechnology, 1:500), polyclonal anti-Bad (Santa Cruz Biotechnology, 1:500), polyclonal anti-5-LO (Cayman Chemicals, 1:500), or polyclonal anti-COX (Cayman Chemicals, 1:500). After washing in Tween-TBS, PVDF sheets were incubated with biotin-conjugated anti-rabbit IgG (1:1000; Santa Cruz Biotechnology) antibody for 1 h and then incubated with Selleck Natural Product Library Methocarbamol horseradish peroxidase-conjugated streptavidin (1:1000; Caltag Laboratories, Burlingame, CA). Immunoreactive proteins were visualized by 3,3′-diaminobenzidine (Sigma) staining and bands were quantified by densitometry using Scion Image Software (Scion Co., MD, USA).

The luminol-enhanced chemiluminescence of human neutrophils was measured using a microplate-reader Spectramax (Molecular Devices, CA, USA), as described previously (Shimoyama et al., 2002). Briefly, cells were stimulated with rHPU (10, 30 or 100 nM) or phorbol 12-miristate 13-acetate (PMA; 30 nM) and ROS production was measured for 60 min. Neutrophils were incubated for 30 min prior to stimulation. In order to measure intra- and extracellular ROS production, CM-H2DCFDA (chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate; λex470 nm, λem529 nm) and lucigenin (bis-N-methylacrydinium nitrate) were used, respectively. The protocol described by (Kujbida et al., 2008) was applied for luminol or lucigenin. For CM-H2DCFDA, neutrophils were incubated with the dye (5 μM) for 15 min at 37 °C prior to stimulation (Espinosa et al., 2009). Male Swiss mice (20–22 g), housed at 22 ± 3 °C with a 12/12 h light/dark cycle were used for the bioassays.