Caspase 1 antibody

Immunoblotting analysis was conducted as previously described [5 (link), 24 (link)]. Antibodies against interleukin (IL)-1β, phospho-myosin light chain (MLC) 2, CK2 and catalase were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against claudin2 were purchased from Invitrogen (Camarillo, CA). Caspase-1 antibody was purchased from Millipore (Billerica, MA). NLRP3 antibody was purchased from Boster Biological (Fremont, CA). IL-18 and perroxiredoxin-4 antibody was purchased from DSHB (University of Iowa, Department of Biology). GAPDH antibody was purchased from GeneTex Inc. (Irvine, CA) while SOD1 antibody was purchased from Santa Cruz Biotech Inc. (Santa Cruz, CA). Density of bands was quantified and then normalized with reference to the GAPDH content.

Western blot analysis was performed using the NOVEX NuPAGE electrophoresis system (Invitrogen, Grand Island, NY) with 1.5 mm 4–12% Bis–Tris gels according to manufacturer’s instructions. Briefly, 25 µg of lung homogeneous was loaded to each well and gels were run at 100V at 4 °C for 2 h in NuPAGE MOPS SDS running buffer under reducing conditions. Proteins were transferred to nitrocellulose membrane at 80V for 60 min at 4 °C. The membrane was then blocked for 1 h at room temperature with 5% BSA in Tween/Tris buffered saline (TTBS; 100 mM Tris base, 1.5 M NaCl adjusted to pH 7.4 with 0.1% Tween 20). Immunoblots were then incubated with caspase-1 antibody (1:1,000, Millipore, Temecula, CA) overnight at 4 °C. The following day, the membrane was washed with TTBS three times, for 20 min each time. A horseradish peroxidase-conjugated goat anti-rabbit antibody (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA), used as the secondary antibody, was applied for 1 h at room temperature. Following this, the membrane was washed with TTBS followed by two 10-min washes with TBS. The blots were developed using a chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). Complete western blots without cropping are shown in Supplementary Fig. 5.