Caspase 5

After the application of cyclic stretch, HPDLCs were washed with ice-cold PBS, scraped from the Bioflex plates and immediately lysed in a lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Protein concentrations of the samples were determined by using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Protein was mixed with an appropriate volume of SDS sampling buffer and separated by SDS-PAGE gel (10%). The protein bands were then transferred onto nitrocellulose membranes by electroblotting. According to the manufacturer's instructions, the membranes were incubated with mouse monoclonal antibody against caspase-1(1:1000, Cell Signaling Technology), caspase-5 (1:1000, Cell Signaling Technology), ASC (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), NLRP1(1:1000, Abcam), NLRP3(1:1000, Abcam), GAPDH (1:10000, Cell Signaling Technology), or rabbit anti-IL-1β (1:1000, Cell Signaling Technology) primary antibodies overnight at 4°C, washed, and then incubated with anti-mouse or anti-rabbit IgG conjugated to HRP (1:5000, Cell Signaling Technology) for 60 min at room temperature. Protein bands were detected by using the ECL SuperSignal reagent (Pierce). Relative band densities of the proteins were measured from scanned films using National Institutes of Health ImageJ Software.

For western blotting, cell or tissue extracts were made by homogenization in lysis buffer. About 20 μg of protein (determined by BCA assay) was boiled with Laemmli buffer. Whole-protein extracts were separated on 6–15% SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). After 1 h blocking procedure, the membranes were probed with primary antibodies in 1:1000 dilution. The antibodies for anti-NLRP3, ASC, caspase-4, caspase-5, pro-GSDMD, cleaved-GSDMD, LC3-II, flotillin-1, HSP70 and GAPDH antibodies were purchased from Cell Signaling (Beverly, MA, USA); anti-pro-caspase-1, cleaved-caspase-1 antibodies were purchased from Proteintech (Rosemont, IL 60018, USA); anti-Lamp-1, CD63 antibodies were purchased from Novus (Centennial CO 80112, USA); anti-TSG-101, p62 antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). After the hybridization, membrane was washed with 1× TBST, and then probed with HRP-conjugated anti-mouse (Biolegend, San Diego, CA, USA) or anti-rabbit secondary antibodies (Jackson ImmunoResearch Lab Inc., West Grove, PA, USA) in 1:10,000 dilution. The immunoreactive proteins were visualized with Immobilon Western chemiluminescence HRP substrate (Merck, USA) and BioMax LightFilm (Eastman Kodak Company, New Heaven, CT, USA) according to the manufacturer’s instructions.