Caspase 8

caspase-8 knockout HeLa cells were transiently transfected using Lipofectamine 2000 according to the manufacturer’s protocol with 19 μg of a pRetroX-Tet-on empty vector (Clontech) and 1 μg of a pcDNA3 vector (ThermoFisher) containing either caspase-8, caspase-8(AAA), or caspase-8(WWW). Media was exchanged after 6 h of transfection, and this time was defined as 0 h after transfection. After the indicated lengths of time, cells in the media were combined with cells that were collected by trypsinization. The cells were then pelleted by centrifugation (6 min, 2000g, 4 °C) and washed with PBS (1 mL) three times. Cell pellets were then resuspended in 50 μL of 1% NP-40 lysis buffer [1% NP-40, 150 mM NaCl, 50 mM TEA, pH 7.4] with Complete Mini protease inhibitor cocktail (Roche Biosciences) and Z-VAD-FMK (Enzo Life Sciences) for 15 min and then centrifuged (10 min, 10000g, 4 °C). The supernatant (soluble cell lysate) was collected, and the protein concentration was determined by BCA assay (Pierce, ThermoScientific). Samples were prepared at a protein concentration of 3 mg mL⁻¹ with 4× loading buffer (8% SDS, 40% glycerol, 0.4% bromophenol blue, 2.8% B-mercaptoethanol, 200 mM Tris-HCl, pH 6.8) and boiled at 5 min at 98 °C. A 40 μg amount of protein was then loaded per lane for SDS-PAGE separation (Criterion TGX 4−20%, Bio-Rad).

Caspase-3 Colorimetric Assay Kit was from BioVision, Inc. (K106-200; Milpitas, CA). Caspase-8 (KHZ0061) and Caspase-9 (KHZ0101) Colorimetric Protease Assay Kits were from Thermo Fisher Scientific (Waltham, MA). The assays were performed following the manufacturers' protocol.^{26 (link)} In brief, cells or tissues were lysed in pre-chilled lysis buffer provided in the kit. Cytosolic extracts (200 μg) in 50 μL lysis buffer and 5 μL chromophore substrate were added to 50 μL reaction buffer. The mixtures were set up in a 96-well plate and assayed for caspase activity in terms of the absorbance of 405 nm after incubation in a microplate reader.