Fluorchem hd2 imager

Protein levels of enriched caveolae were determined by the Bradford protein assay (BioRad, Cat # 5000001). Quantities of 1, 2 and 5 µg of protein were ran through 15% acrylamide gel with a 5% stacking gel for cav-1, ER-α and Her-2/neu, respectively, to determine protein expression by Western protein blotting. The separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, blocked overnight in 5% skim milk powder and incubated for 2 h at room temperature with either caveolin-1 antibody (Santa Cruz, Cat # sc-894) diluted 1:500 or ER-α antibody (Santa Cruz, Cat # sc-7207) diluted 1:200 or Her2 neu antibody (Santa Cruz, Cat # sc-101695) diluted 1:200. All primary antibodies were followed by goat anti-rabbit IgG HRP (Santa Cruz, Cat # sc-2030) diluted 1:1000 for 1 h at RT. Protein bands were detected by Western Lightening Plus ECL (Perkin Elmer, Cat # NEL 103001EA) and visualized on a FluorChem HD2 imager (Cell Biosciences, Santa Clara, California, USA). Bands were quantified using AlphaView imaging software (Version 3.1.1.0).

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.