Western blot quant hrp substrate

The expression of IL-1β in supernatants was analyzed using SDS-PAGE. After transfer onto PVDF membranes, non-specific antibody binding was blocked for 1 h at room temperature using 5% skim milk. Membranes were then incubated for 1 h at room temperature or 24 h at 4C with anti-human IL-1β antibody (R&D systems), anti-signal transducer and activator of transcription 3 (STAT3) antibody and anti-phospho-STAT3 antibody (Cell Signaling Technology, Inc., Boston, MA), anti-caspase-1 antibody (Cell Signaling) followed by incubation for 1 h with secondary antibody conjugated horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories, Inc., PA). Immunoreactive bands were visualized by Western BLoT Quant HRP Substrate (Takara Bio Inc., Shiga, Japan).

Strain UMI-01 was cultured in the minimum salt medium containing 2% (w/v) glucose or 1% (w/v) alginate as the sole carbon source at 25 °C. When the OD₆₀₀ of the culture medium was between 0.6 and 0.8, the cells were harvested by centrifugation at 12,000g for 15 min at 4 °C, and soluble proteins were obtained using BugBuster Master Mix (Merck Millipore, Burlington, MA, USA) according to the manufacturer’s protocol. Each sample (5 µg) was subjected to SDS-PAGE and electroblotted onto a ClearTrans nitrocellulose membrane (FUJIFILM Wako Pure Chemical Corporation). The anti-FlRed, -FlKin, -FlAld, -FlDet, and -FlDeg antibodies were raised in rabbits against the synthetic peptides CKGAERVAELAAEGI, CYGLNEEPLDNQRAL, CSKLIKPESDGNFDL, CLVMDSRKDPRAASA, and CDTAQPNRTPLPKSD, respectively, using keyhole limpet hemocyanin as a carrier protein (Eurofins Genomics, Tokyo, Japan). Each antibody was purified by affinity chromatography using an immobilized antigen column (Cellufine Formyl, JNC Corporation, Tokyo, Japan) and used as the primary antibody. A horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich) was used as the secondary antibody, and signals were detected using Western BLoT Quant HRP Substrate (TaKaRa).