Anti caspase 8 1c12

Whole-cell lysates were generated by the addition of 2× Laemmli buffer containing 10% β-mercaptoethanol to the cell pellets. Cell lysates were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Membranes were blotted with the following antibodies: anti-caspase-8 (1C12; Cell Signaling), anti-caspase-3 (8G10; Cell Signaling), anti-PCNA (D3H8P; Cell Signaling), and anti-β-actin (AC-15; Sigma-Aldrich). Peroxidase-conjugated secondary antibodies were used (BioLegend). Membranes were developed using enhanced chemiluminescence (ECL) (Thermo Scientific) and detected using a Nikon camera as previously described (82 (link)). Quantification analysis of blots was performed using ImageJ, and the β-actin signal was used as a control for normalization. Values are expressed as a percentage of the normalized signal from the mock-infected group.

Aur and pan caspase inhibitor z-VAD-FMK were obtained from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA) and dissolved in DMSO at a stock concentration of 10 mM, aliquoted and stored at −80°C. DSF was acquired from Sigma-Aldrich (St. Louis, MO). MTS assay (CellTiter 96 Aqueous One Solution reagent) was purchased from Promega Corporation (Madison, WI, USA). PI and Annexin V-FITC apoptosis Detection Kit, DCFH-DA and cell apoptosis Rhodamine 123 Detection Kit were purchased from Keygen Company (Nanjing, China). Antibodies (Abs) used in this study were purchased from following sources: anti-ubiquitin (P4D1) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-PARP, anti-BIP (C50B12), eIF2α, Phospho-eIF2α(Ser51), anti-IRE1α (14C10), anti-caspase-3 (8G10), anti-Bcl-2 (50E3), anti-Bcl-xl (54H6), anti-CHOP (L63F7), anti-HSP70, anti-HSP90, anti-caspase-8 (1C12), and anti-caspase-9 (C9) (Cell Signaling Technology, Beverly, MA, USA); anti-cleaved caspase-8 (Cleaved Asp384) (Assay biotechnology Company, Inc); anti-cleaved caspase-9 p35 (D315), anti-cleaved caspase-3 (p17), anti-Bim (Y36), anti-ATF4 (R239), anti-Noxa (EPR9735 [B]), and anti-GAPDH (Bioworld Technology, Inc).

Recombinant human TRAIL and anti-His were obtained from R&D Systems (Minneapolis, MN). MG132, propidium iodide (PI), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and anti-Flag-HRP were purchased from Sigma (St. Louis, MO). Rabbit anti-DTX1 polyclonal antiserum against GST-Deltex1 was generated as described^{9 (link)}. The following antibodies were obtained from Santa Cruz Biotech (Santa Cruz, CA): anti-caspase-3 (H-277), anti-DR4 (H130), anti-DR5 (N-19), anti-Mcl-1 (S-19), and anti-Bcl-2 (N-19). Annexin V-Cy5 was obtained from Biovision (Mountain View, CA). Anti-Myc (9B11), anti-caspase-8 (1C12), and anti-active caspase-3 (D175) were purchased from Cell Signaling (Beverly, MA). Anti-actin (clone C4) and anti-β-tubulin (clone AA2) were purchased from Millipore (Temecula, CA). Anti-human FLIP mAb (NF6) was purchased from AdipoGen (San Diego, CA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. WesternBright ECL HRP substrate was obtained from Advansta Corporation (Menlo Park, CA). Dapi-Fluoromount-G^TM was obtained from SouthernBiotech (Birmingham, AL). Protein G Mag Sepharose^TM Xtra was obtained from GE Healthcare (Piscataway, NJ).