Mouse anti caspase 8

Whole protein extracts were obtained by washing the cells with ice cold PBS and adding lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol) supplemented with 1X cOmplete Proteinase Inhibitor Cocktail and 1X PhosSTOP Phosphatase Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany). Cells were detached by scraping. The lysates were incubated for 20 minutes at 4 °C and afterwards centrifuged at 14000 rpm for 30 minutes at 4 °C. Western blot analysis was performed as described elsewhere^{18 (link)}. The following antibodies were used: rabbit anti-phospho AKT (S473), rabbit anti-AKT, mouse anti-phospho ERK1/2 (T202/Y204), rabbit anti-phopsho JNK (T183/Y185), mouse anti-phospho IκBα (S32/S36), rabbit anti-IκBα, rabbit anti-caspase-3 (Cell signaling, Cambridge, UK), mouse anti-JNK (BD, Heidelberg, Germany), mouse anti-caspase-8 (Alexis, Grünberg, Germany), rabbit anti-ERK1/2, mouse anti-β-actin (Sigma-Aldrich, Munich, Germany), mouse anti-caspase-8 (Enzo LifeSciences, Lörrach, Germany) and mouse anti-α-tubulin (Calbiochem/EMD Millipore, Darmstadt, Germany). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were from Santa Cruz Biotechnology (Heidelberg, Germany). Full length blots are provided in the Supplementary Appendix.

Protein measurements were carried out using the Pierce BCA Protein Assay Kit (Thermo Scientific) in accordance with the manufacturer’s instructions. Samples were mixed with Laemmli’s loading buffer, boiled for 5 min, and subjected to SDS-PAGE (12%) followed by blotting onto nitrocellulose membranes for 30 min at 25 V using the Mini Trans-Blot Cell (Bio-Rad). Membranes were blocked for 1 hr with 5% non-fat milk in TBS at room temperature and subsequently probed overnight at 4°C with the primary antibody (1:1000). The following primary antibodies were used: rabbit anti-vinculin, rabbit anti-hexokinase-II, rabbit anti-Na⁺/K⁺-ATPase, rabbit anti-cleaved caspase-3 (all from Cell Signaling), mouse anti-caspase-2, mouse anti-caspase-3, mouse anti-PARP1, mouse anti-cytochrome c (all from BD Transduction Lab), mouse anti-lamin B, rabbit anti-Endonuclease G, mouse anti-AIF (all from Santa Cruz Biotech), mouse anti-caspase-8 (Enzo Life Science), rabbit anti-GAPDH (Trevigen), and rabbit anti-ERp29 (kindly provided by Dr. S. Mkrtchian, Karolinska Institutet). After four times washes in TBST (0.05% Tween-20 in TBS), membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies purchased from Cell Signaling (1:4000) for 1 hr at room temperature. Blots were developed using ECL (Amersham Biosciences) and documented using Chemi-Doc (Bio-Rad).

Western blot analysis was performed as described previously [25 (link)] using the following antibodies: mouse anti-XIAP, mouse anti-RIP1, mouse anti-FADD from BD Biosciences (Heidelberg, Germany), goat anti-cIAP1 from R&D Systems (Wiesbaden, Germany), rat anti-cIAP2, mouse anti-caspase 8 from Enzo Life Sciences (Lörrach, Germany), rabbit anti-caspase-3 from Cell Signaling Technology (Danvers, USA), rabbit anti-caspase 8 from Epitomics (Burlingame, USA) and mouse anti-GAPDH from HyTest (Turku, Finnland) followed by goat-anti-mouse IgG or goat-anti-rabbit IgG conjugated to horseradish peroxidase purchased from Santa Cruz Biotechnology, Inc. (Dallas, USA). Enhanced chemiluminescence was used for detection from Amersham Bioscience (Freiburg, Germany). Further, donkey anti-mouse IgG, donkey anti-rabbit IgG or donkey anti-goat IgG labeled with IRDye infrared dyes were used for fluorescence detection at 700 nm 800 nm (LI-COR Biotechnology, Bad Homburg, Germany). Immunoprecipitation was performed as previously described [26 (link)] using RIPA buffer or NP-40 buffer for cell lysis.

Western blot analysis was performed as described previously [44 (link)] using the following antibodies: Mouse anti-cFLIP (Enzo Life Sciences, Lörrach, Germany), goat anti-cIAP1 (R&D Systems, Wiesbaden, Germany), rat anti-cIAP2 (Enzo Life Sciences), mouse anti-XIAP (BD Biosciences, Heidelberg, Germany), rabbit anti-NIK (Cell Signaling Technologies, Beverly, MA USA), mouse anti-p100/p52 (Merck Millipore, Darmstadt, Germany), rabbit anti-p62 (MBL International, Woburn, MA, USA), mouse anti-Noxa (Enzo Life Sciences), rabbit anti-Mcl-1 (Enzo Life Sciences), mouse anti-caspase-8 (Enzo Life Sciences), rabbit anti-caspase-3 (Cell Signaling Technologies), rabbit anti-caspase-9 (Cell Signaling Technologies), mouse anti-β-actin (Sigma-Aldrich), mouse anti-GAPDH (HyTest, Turku, Finland), mouse anti-tubulin (Calbiochem, Merck Millipore, Darmstadt, Germany). Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for enhanced chemiluminescence detection (Amersham Bioscience, Freiburg, Germany). Alternatively, secondary antibodies labeled with IRDye infrared dyes were used for fluorescence detection (Odyssey Imaging System, LI-COR Bioscience, Bad Homburg, Germany). All Western blots shown are representative of two or three independent experiments.