0.2 μm pore size nitrocellulose membrane

Ending the culture, cells were harvested, washed in PBS, and lysed in RIPA buffer containing protease inhibitor (Complete Mini Protease Inhibitor Cocktail Tablet, Roche). Cellular protein extracts were separated by SDS-PAGE and transferred to 0.2 μm pore-size nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA) with 25 mm Tris-base (pH 8.0) containing 150 mm glycine and 20% (volume/volume) methanol as previously described [28 (link)]. Membranes were incubated with antibodies to phospho-p44/42 MAPK (ERK1/2), phospho-nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65), phospho-apoptosis signal-regulating kinase 1 (ASK1) (Ser967) (from Cell Signaling Technology), rabbit polyclonal anti-iNOS (Calbiochem Merck), and anti-caspase-1 (clone 5B10) (BioLegend). Protein bands were detected by incubating with horseradish peroxidase-labeled antibodies and visualized with enhanced chemiluminescence reagent (Advantas) using an ImageQuant Las-4000 mini (GE Healthcare Life Science). Band densities were analyzed by densitometry using the online IMAGEJ 1.39c software (National Institutes of Health) (http://rsb.info.nih.gov/ij/index.html) as described by Luke Miller (http://www.lukemiller.org/journal/2007/08/quantifying-western-blots-without.html). Samples were normalized using tubulin as loading control.

After SDS‐PAGE, the gels were processed for immunoblotting using Trans‐Blot Turbo Transfer System (BioRad, Hercules, California, USA) and 0.2 μm pore‐size nitrocellulose membranes (BioRad). Immunoblot detection was done with Western Lightning ECL Pro (Perkin Elmer, Winter St. Waltham, Massachusetts, USA), and a ChemiDoc XRS + system (BioRad) or exposure to Amersham Hyperfilm ECL (GE Healthcare).

SO and CHA powders were purchased from Sigma-Aldrich Co., LLC (Tokyo, Japan). Horseradish peroxidase (HRP)-labeled goat anti-rabbit polyclonal IgG (H + L) antibodies were obtained from Funakoshi Co., Ltd. (Tokyo, Japan). Commercial serum and urine samples of healthy volunteers were obtained from Cosmo Bio Co., Ltd. (Tokyo, Japan). Bio-Safe Coomassie stain, Clarity Western ECL substrate kits, Precision Plus Protein Dual Color Standards, Trans-Blot Transfer Packs, and 0.2 μm pore-size nitrocellulose membranes were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). A 96-well ELISA plate H was purchased from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan). An HRP labeling kit was obtained from Dojindo Laboratories (Kumamoto, Japan). Blocking One and dimethyl sulfoxide (DMSO) were purchased from Nacalai Tesque, Inc (Kyoto, Japan). The Prominence LC-2010 HPLC system and the reversed-phase chromatography (RPC) column Shim-pack GIST C18-AQ were purchased from Shimadzu Corporation (Kyoto, Japan). Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), o-phenylenediamine dihydrochloride (OPD) tablets, 2-mercaptoethanol (2-ME), and all other reagents were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) and Wakenyaku Co., Ltd. (Kyoto, Japan).

Total proteins were extracted from 50mg of frozen shoots using 200 μl of solution [50mM TRIS, 2mM EDTA, 10% (v/v) glycerol, and 10mM 2-merceptoethonal, pH 8.0]. The homogenates were centrifuged at 16 099 g for 3min at 4 °C. The supernatant was analysed by western blot analysis. The total protein concentration was determined by a nanodrop 2000 spectrophotometer (Thermo Scientific). A 10 μg aliquot of crude proteins was separated on 12% Criterion XT Bis-Tris gels (Bio-Rad), electrophoretically transferred to 0.2 μm pore-size nitrocellulose membranes (Bio-Rad), and blocked with PBST [8mM K₂HPO₄, 3.9mM KH₂PO₄, 150mM NaCl, and 0.05% (v/v) Tween-20, pH 7.2] containing 5% skim milk. The blocked membrane was then incubated with 20 μg of anti-GS serum (rabbit IgG) in 20ml of PBST at 4 ºC overnight. After several washes with PBST, the membrane was incubated at room temperature for 2h with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10 000) (Thermo Scientific), and the immune complexes were detected using chemiluminescence reagents.