Anti bid

Cells were seeded in supplemented media at a determined density for Hepa1-6 (0.125M cells/cm²) and Dt81Hepa1-6 (0.250M cells/cm²) for 7h on 10cm dishes coated with COL1. Media was then changed overnight for serum-free DMEM. Cells were then harvested and subjected to a 12% SDS-polyacrylamide gel electrophoresis and blotted as described [20 (link)]. Membranes were blocked and probed with the following antibodies: anti-beta-catenin, anti-ERK1/2 and anti-phosphospecific ERK1/2, anti-AKT and anti-phosphospecific AKT, anti-Bad and anti-phosphospecific Bad (all from Cell Signaling, Pickering, On, Canada), anti-Bcl-XL (Transduction Laboratories, Mississauga, On, Canada), anti-Bak (Upstate, Lake placid, NY, USA), anti-Bid (R&D system, Minneapolis, MN, USA) and anti-actin kit (Calbiochem, Merck KGaA, Darmstadt, Germany) for 2h in PBST containing 1% milk at room temperature. Membranes were washed and then incubated with HRP-conjugated secondary anti-rabbit IgG, anti-mouse IgG (both from BD Pharmingen, San Diego, California, USA), anti-rat IgA (Cedarlane labs, Burlington, On, Canada) or anti-mouse IgM (from anti-actin kit) antibodies at room temperature in PBST containing 5% milk during 1h. After extensive washes in PBST, bound peroxidase was detected with enhanced chemiluminescence blotting substrate (Perkin-Elmer, Woodbridge, On, Canada), according to the manufacturer’s instructions.

Intact cells or membrane fraction (pelleted by centrifuging permeabilized cell suspensions at 10,000 × g for 5 min) were lysed in cold radioimmunoprecipitation assay buffer, (150 mM NaCl, 1.0% (vol/vol) Octylphenoxypolyethoxyethanol, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulphate, and 50 mM Tris (pH 8.0; Sigma)), supplemented with 1 μg/ml protein inhibitors (leupeptin, antipain, and pepstatin) and 1 mM phenylmethylsulfonyl fluoride. Lysed samples and cytosol fractions were used for immunoblotting. Western blot was performed based on instructions of LI-COR (LI-COR Corporation, Nebraska, USA). Primary antibodies used were Anti-BAK (no. 06-536; Millipore) Anti-cyto c (no. 556433; BD Bioscience), Anti-HA (no. 9110; Abcam), Anti-mtHsp70 (no. MA3-028; Thermo Scientific), and Anti-prohibitin (no. ab28172; Abcam), anti-Bax (N-20, sc-493, SantaCruz), anti-MCL-1 (ADI-AAP-240, Enzo life sciences), anti-Bid (no. AF860, R&D systems), anti-Bim (no.2933, Cell Signaling), anti-Bcl-xL (no. 610211, BD transduction), anti-Actin (no.612656, BD transduction), anti-Calnexin (no. ADI-SPA-860, Enzo Life sciences). Detection of bands was performed on a LI-COR Odyssey scanner. ImageJ was used for quantification of the bands.

Recombinant expression and purification of LFn-FlaA was performed as previously described [19 (link)]. B. anthracis protective antigen (PA) and lethal factor (LF) were acquired from List Biologicals. TMRM (T668), Lysotracker (L7528), Sytox Green (S7020), Mitotracker (M7512), Fluo4 (F14217), Pluronic F-127 (P6867), Hoechst 33342 Trihydrochloride Trihydrate (H1399), PI (P3566) and CTB coupled to Alexa 594 (C22842) or 647 (C34778) were purchased from Thermo Scientific. PI solution (556463) and Annexin-V–FITC (556419) were from BD Biosciences. The antibodies used in the study were anti-Caspase-1 (AG-20B-0042-C10, Adipogen), anti-Cathepsin B (31718S, Cell Signaling Technology), anti-Cytochrome c (11940S, Cell Signaling Technology), anti-HMGB1 (ab18256, Abcam), anti-Caspase-3 (9662S, Cell Signaling Technology) and anti-β-Actin-HRP (sc-47778, Santa Cruz Biotechnology), anti-BID (AF860, R&D Systems). Horseradish peroxidase (HRP)-conjugated secondary antibodies were acquired from Jackson Immunoresearch Laboratories and enhanced chemiluminescence solution was from Thermo Scientific. Punicalagin (P0023) was from Sigma Aldrich and the CytoTox 96 Non-Radioactive Cytotoxicity Assay (G1780) and FugeneHD Transfection Reagent were purchased from Promega. Y27632 and (−)-blebbistatin were acquired from Selleckchem. Pam3csk4 (tlrl-pms) and LPS-SM (tlrl-smlps) were acquired from Invivogen.