Caspase 11

HEK293 cells were transfected with mammalian expression constructs for myc-tagged pro-caspase-4 or Flag-tagged pro-caspase11 in six-well plates using Lipofectamine 2000 (Invitrogen). Thirty-six hours post transfection, cells were lysed in 50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM EDTA, 10% glycerol, and 1% Triton X-100, supplemented with protease inhibitors (Roche) for 30 min. For LPS binding, total cell lysates (TCLs) were incubated with 1 μg ultra-pure biotinylated E. coli LPS (0111:B4; Invivogen) in the presence or absence of 100 μg oxPAPC (Invivogen) or unlabeled LPS for 2 h at room temperature and purified with immobilized NeutrAvidin (Thermo Scientific), washed three times in binding buffer, eluted by Laemmli buffer, and analyzed by immunoblotting using anti -c-myc (Santa Cruz) and anti-Flag antibodies (Sigma), and horseradish peroxidase-conjugated secondary antibodies, ECL detection (Thermo Scientific), and image acquisition (Ultralum). TCLs (5%) were also analyzed as indicated. For oxPAPC binding, TCL were incubated with 10 μg oxPAPC in the presence or absence of 100 μg LPS for 2 h and pulled down by 1 μg biotinylated E06 antibody, immobilized onto NeutrAvidin beads, then analyzed as described above. For endogenous caspase binding, BMDMs were primed with poly(I:C) for 16 h and TCLs were analyzed as described above for caspase-11 (Sigma).