Sc 81656

Tissue homogenates were resuspended in 1 × lysis buffer (1% NP-40, 100 mM Tris, pH 8.0, and 150 mM NaCl) containing a protease inhibitor mixture (Roche) and shaken 60 min at 4 °C. Samples were centrifuged and lysate was collected. Protein concentration was quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific). Tissue lysates were mixed with 2 × Laemmli sample buffer and incubated at 100 °C for 5 min and equal amounts of protein were loaded into a 4–15% polyacrylamide gel for SDS/PAGE and transferred onto a nitrocellulose membrane (Bio-Rad). Membranes were incubated with the following primary antibodies: anti-caspase-1 (p20) (Casper-1, AdipoGen), anti-caspase-3 (#9662, Cell Signaling Technology), anti-caspase-7 (#9492, Cell Signaling Technology), anti-caspase-8 (#8592, Cell Signaling Technology), anti-caspase-8 (sc-81656, Santa Cruz Biotechnology), anti-Lipocalin-2 (AF1857-SP, R&D Systems) and anti-β-actin (sc-8432, Santa Cruz Biotechnology). Membranes were then probed with anti-mouse, anti-rabbit (Thermo Fisher Scientific) or anti-goat (Promega) secondary antibodies conjugated to horseradish peroxidase, and antibody complex were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).

Coimmunoprecipitation was performed using a Thermo Scientific Pierce Coimmunoprecipitation Kit (Thermo Scientific, Rockford, AL, USA) as previously described 32 (link), 33 (link). The following antibodies were used: anti-FADD (14906-1-AP; Proteintech), anti-caspase-8 (AF6442; Affinity; sc-81656; Santa Cruz), anti-RIPK1 (A7414; ABclonal), anti-Drp1 (ab156951; Abcam; 8570S; Cell Signaling Technology), anti-Fis1 (A5821; ABclonal) and anti-TOMM20 (ab186735, ab283317, ab78547; Abcam). In addition, normal IgG was added, and the samples were incubated at 4 °C overnight on a rotator. Then, 25 µl of G/A agarose beads was added to 200 µl of lysate, and the mixture was incubated for 3 h with gentle agitation at room temperature according to the manufacturer's protocol. The beads were rinsed, boiled and then removed by centrifugation at 14,000 × g for 12 min, after which the final samples were analyzed via western blotting.