Ecl luminol reagent

A total of 10⁷ BMDMs were seeded per well, primed with ultrapure LPS (500 ng/ml) for 4 h and then infected with L.g.− or L.g.+ for 48 h. The supernatants were collected and precipitated with 50% trichloroacetic acid (TCA) and acetone. After their clarification by centrifugation, the cells were lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, and 0.1% SDS) containing protease inhibitor cocktail (Roche). The lysates and supernatants were resuspended in Laemmli buffer, boiled, resolved by SDS-PAGE and transferred (Semidry Transfer Cell, Bio-Rad) to a nitrocellulose membrane (GE Healthcare). The membranes were blocked in Tris-buffered saline (TBS) with 0.01% Tween-20 and 5% non-fat dry milk. The rat anti-Casp1 p20 monoclonal antibody clone 4B4 (Genentech) (1:500), goat anti-IL-1β p17 subunit (Sigma-Aldrich, 1:250) and specific anti-rat or anti-goat horseradish peroxidase-conjugated antibodies (1:3000; KPL) were diluted in blocking buffer for the incubations. The ECL luminol reagent (GE Healthcare) was used for the antibody detection.

To assess caspase-8 activation, BMDMs were infected with fliI^- or flaA^- for 8 hours, and the activity of caspase-8 was measured using the Caspase-8 Glo Assay (Promega) according to manufacturer´s recommendations. To evaluate caspase-8 activation by western blot analysis, 1 X 10⁷ cells were infected at a MOI of 10 and lysed 8 hours after infection in RIPA buffer with protease inhibitor, as previously described. The lysates were suspended in 4X Laemmli buffer, boiled for 5 minutes, resolved by 15% SDS PAGE and transferred to 0.22-μm nitrocellulose membranes. The membranes were blocked in Tris-buffered saline (TBS) with 0.01% Tween-20 and 5% non-fat dry milk or for 1 hour. The mouse anti-caspase-8 (Enzo– 1G12) and anti-rabbit peroxidase-conjugated antibody (KPL; 1:3000) were diluted in blocking buffer for the incubations (overnight for anti-cleaved caspase-8 and 1 hour for the secondary antibody). ECL luminol reagent (GE Healthcare) was used for antibody detection.