For standard Western blotting analysis (cell extracts only), 0.5 × 106 cells were lysed in 75 μl of NP-40 buffer [10 mM tris (pH 7.5), 150 mM NaCl, 1% NP-40, 2.5 mM MgCl2, and 0.5 mM CaCl2]. For Western blotting analysis of the caspase-11 p26 fragment, cell culture medium and extracts from LPS-stimulated iMACs were prepared as reported previously (32). Briefly, ~8.0 × 105 cells were electroporated as described earlier with the Neon Transfection System and then were incubated in 75 μl of Opti-MEM I medium for 2 hours. Cell culture medium was collected, and the cells were lysed in NP-40 buffer with 1× cOmplete Protease Inhibitor (Roche Applied Science) and then combined with the cell culture medium with additional protease inhibitor.

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