Novex nupage electrophoresis system

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 mg of lung homogeneous was loaded to each well and gels were run at 100 V at 4 °C for 2 h in NuPAGE MOPS SDS running buffer under reducing conditions. Proteins were transferred to nitrocellulose membrane at 80 V for 60 min at 4 °C. The membrane was then blocked for 1h 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 myc-tag antibody 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).

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.