Caspase 8

To characterize the expression of CFP-linker-YFP probe, the expressed protein was isolated using anti-CFP/YFP antibody. Briefly, 1.2×10⁶ MB231_CFP-YFP cells were harvested and lysed in 1 ml of Pierce immunoprecipitation lysis buffer. 25 µl of anti-CFP/YFP antibody conjugated to sepharose (Abcam, Cambridge, MA) was rotated with debris-free cell lysate for 3 h at 4°C. After washing with lysis buffer, bound CFP- linker-YFP FRET probe was eluted with 50 mM Tris buffer (pH 8.5) containing 1 M NaCl. Immediately after elution the protein was diluted with Tris buffer to yield a final concentration of NaCl of 300 mM. Protein purity was visualized by SDS-PAGE and Coomassie blue staining and by immunoblot analysis using antibodies specific for CFP and YFP (described above).
For in vitro FRET assays, immuno-purified CFP-linker-YFP FRET probe was incubated with active caspase 3, caspase 8, or caspase 2 (R & D systems, Minneapolis, MN) for 3 h at 25°C in PBS buffer containing 1 mM DTT using black 96 well microplates. As a control Z-VAD was also added to some reactions. FRET was measured as described above.

For western blotting, 50 μg proteins from cell lysates, as determined by the Micro BCA Protein Assay (Merck Millipore, Billerica, MA, USA), were loaded on 10% SDS-PAGE gels, electrophoresed, and transferred onto polyvinylidene difluoride membrane (Merck Millipore). The membranes were probed for caspase-3 (R&D Systems, Minneapolis, MN, USA; 1 : 500 dilution), caspase-6 (R&D Systems, 1 : 800 dilution), and caspase-8 (R&D Systems, 1 : 400 dilution). Immunodetected protein bands were quantified with Image J software (NIH, Bethesda, MD, USA).

CSF1, GM-CSF, IL-4, Q-VD-OPh and Z-VAD-FMK were obtained from R&D Systems (Minneapolis, MN, USA). Ac-YVAD-cmk from InvivoGen (San Diego, CA, USA), staurosporine and cleaved caspase-3 blocking peptide (#1050) from Cell Signaling (Danvers, MA, USA), IDN-6556 from MedChemtronica (Stockholm, Sweden), Tiron, TEMPO and Mito-TEMPO from Santa Cruz (Dallas, TX, USA). We used antibodies to calreticulin (#ab22683, Abcam, Cambridge, MA, USA), active caspase-3 (#9664, Cell Signaling), caspase-3 (#7148, Santa Cruz), caspase-3 (#9662, Cell Signaling), caspase-7 (#9492, Cell Signaling), caspase-7 (#sc-81654, Santa Cruz), caspase-8 (#9746, Cell Signaling), caspase-8 (#AF705, R&D Systems), DIABLO (#2409, ProSci, Poway, CA, USA), FADD (#2782, Cell Signaling), GST (#sc-138, Santa Cruz), γ-H2AX (#05-636, Merck Millipore, Billerica, MA, USA), HSC70 (#ADI-SPA-816, Enzo Life Sciences, Farmingdale, NY, USA), mtHSP70 (#MA3-028, Thermo Fisher Scientific, Waltham, MA, USA), LC3 (#M152-3, MBL International, Woburn, MA, USA), NOX2 (#sc-130543, Santa Cruz), p47^PHOX (#4312, Cell Signaling), TOM20 (#sc-11415, Santa Cruz).