Anti mouse il1β

In order to detect the activated form of IL-1β, caspase-1 and IL-18 from NaOCl-treated AMJ2-C11 cells, Culture supernatants concentrated using Amicon Ultra Centrifugal Filters for Protein Purification and Concentration (Millipore, Billerica, MA, USA) and cell lysates were used. Sodium dodecyl sulfate (SDS) and β-mercaptoethanol were added to all samples, and final sample protein concentrations were adjusted by adding more lysis buffer. Proteins (20 µg/well) from culture supernatants and cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and blotted onto PVDF membranes. The membranes were then probed with anti-mouse IL1β (Abcam, Cambridge, MA, USA), caspase-1 (Santa Cruz Biotech, Santa Cruz, CA, USA) and IL-18 (Santa Cruz Biotech) antibodies prior to incubation with HRP-conjugated secondary antibodies. The blots were developed using the Super-Signal West Dura chemoluminiscence system (Pierce, Perbio Science, Helsingborg, Sweden) according to the manufacturer's instructions. The bands representative were detected by FluorChem ™ HD2 Imager (Cell Biosciences). TSLP expression was analyzed with western blot in the cell lysates from NaOCl-treated A549 cells.

Protein extraction was performed in accordance with the protocol of Chomczynski [26 ]. Briefly, protein precipitation from the organic phase was prepared by adding isopropanol. Precipitate was washed with ethanol and dissolved in 0.5% sodium dodecyl sulfate solution. Quantifications and determination of protein purity were performed with a NanoDrop Spectrophotometer (ThermoScientific NanoDrop Technologies). Total protein (30 μg) was mixed with 4X Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) and loaded on 4% to 15% Mini-PROTEAN TGX™ Precast Gel (Bio-Rad Laboratories) for electrophoresis. Transfer was done with polyvinylidene fluoride (PVDF) transfer membranes (TRANS-Blot® Turbo™ System, Bio-Rad Laboratories). The membranes were blocked overnight at 4°C followed by primary antibodies—rabbit polyclonal anti-mouse IL-1β (Abcam), rabbit polyclonal anti-mouse IL-6 (Abcam), and rabbit polyclonal anti-mouse GAPDH (Santa Cruz Biotechnology) in blocking buffer overnight at 4°C—and with secondary antibody: horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories) for 1 hour. Immunoblotting bands were visualized by Immun-Star™ WesternC™ Chemiluminescence Kits, and a Gel Doc™ EZ System (Bio-Rad Laboratories) was used for imaging and protein-band assay.