Caspase 8

For western blot analysis, Caco-2 monolayers were grown on 6-well cell-culture plates (corning) until 100% confluency. Monolayers were then incubated with 1 × 10⁸ parasites/mL for 6 and 12 h, respectively, washed with PBS twice, and then scraped and collected. Monolayers were then incubated with RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Pierce). Lysed samples were centrifuged at 21,000 rpm at 4°C for 30 minutes. Protein concentration of the supernatant was determined with the D_C Protein Assay (Bio-Rad Laboratories). SDS-PAGE gels (12% and 15% Tris-HCL Ready-Gels; Bio-Rad Laboratories) were used to separate total proteins. Proteins were then transferred using a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore). 5% of nonfat dry milk in % TBS-T was then used to block the membranes. After blocking, membranes were incubated with primary antibodies against, caspase 3, caspase 8, caspase 9, or ZO-1 (1 : 1000; Sigma) overnight at 4°C. After incubation with primary antibodies, membranes were washed and incubated with HRP-tagged secondary antibodies. Bands were detected using Amersham ECL Plus western blotting detection system (GE Healthcare). Autoradiographic films (Kodak) were then exposed to the membranes and developed on X-ray film processor SRX-101A (Konica Minolta).

Lanatoside C (lana.C), rottlerin, Ro318220, Gö6983 were purchased from Sigma Chemical Co. (St Louis, MO, USA). The above drugs were all dissolved in dimethylsulfoxide (DMSO). The non-conjugated primary antibodies were against C23 (nucleolin, Sigma Chemical Co., St Louis, USA), BID, caspase-8, caspase-9, PKCδ (Thr505), PKCδ, caveolin, phoshpo-p44/42 MAP kinas (Thr202/Tyr204), p44/42 MAP kinas (Thr202/Tyr204), phosho-SAPK/JNK (Thr183/Tyr185), p38 MAP kinase, phosho-mTOR, phosphor-p70 S6 kinase (Thr421/Ser424), phosphor-4E-BP1 (Thr37/46), phosho-eIF4E (Ser209), AKT (Cell signaling Technology, Beverly, MA, USA), AIF, Bcl-xl, Mcl-1, α-tubulin, PARP-1/2 (Santa Cruz Biotechnology Corp., Santa Cruz, CA, USA), caspase-2, caspase-6, caspase-7 (BD Biosciences, San Jose, CA, USA), phosho-AKT (Ser473, Epitomics Inc., Burlingame, CA, USA), caspase-3 (Imgenex, San Diego, CA, USA), β-actin (Millipore, Temecula, CA, USA). The secondary horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). z-VAD-fmk was purchased from R&D system (Minneapolis, MN, USA).

The protein sample was added to the corresponding sodium dodecyl sulfate (SDS) gel sample buffer, boiled, denaturalized for 5 minutes, before SDS-polyacrylamide gel electrophoresis, and then transferred onto membranes. The membranes were blocked in phosphate buffered saline with Tween (PBST) containing 5% defatted milk powder at room temperature for 1 hour, washed three times with PBST, and incubated with primary antibodies for NF-κB, TNF-α, IL-1β, IL-4, IL-6, IL-10, SOD-1, MDA5, IREa, Bcl-2, Bax, Caspase3, Caspase8, DDAH1, ADMA and p38(Sigma, USA) overnight at 4°C. Subsequently, the blot was washed and incubated with goat antimouse IgG conjugated to peroxidase(Sigma, USA). Antibody binding was detected by chemiluminescence staining using an ECL detected kit (Bio-Rad, USA). The density of each band was quantified by densitometry using Bandscan 5.0 software.

Cells were lysed in whole cell lysis buffer (100 mM Tris‐HCl pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, 0.1% (w/v) bromophenol blue, 2.5% (v/v) β‐mercaptoethanol). Proteins were separated by SDS‐PAGE and transferred to nitrocellulose membranes (GE Healthcare, Munich, Germany), which were then blocked in 5% (w/v) non‐fat milk dissolved in PBS containing 0.1% (v/v) Tween 20 (Sigma‐Aldrich, P5927). Membranes were probed with specific antibodies against: caspase‐9 (Cell Signaling Technology (CST), Danvers, MA, USA, #9508), FADD (Santa Cruz, Dallas, USA, sc‐6036), cleaved caspase‐8 (CST, #8592), caspase‐8 (CST, #4790), caspase‐3 (CST, #9662), Atg5 (CST, #12994), light‐chain 3 (LC3) (Sigma‐Aldrich, L7543), p‐eIF2α (CST, #3398), eIF2α (CST, #5324), ATF4 (CST, #9508), CHOP (CST, #2895), FLAG (Sigma, F1084) and actin (Sigma‐Aldrich, A2066). Horseradish peroxidase (HRP)–conjugated antibodies were from Jackson ImmunoResearch, Ely, UK: anti‐rabbit (111–035–003), anti‐mouse (115–035–003) and anti‐goat (705–035–003). The signal was visualized using enhanced chemiluminescence reagent (PerkinElmer, Waltham, Massachusetts, USA, NEL102001EA) or ImmunoCruz Luminol Reagent (Santa Cruz, sc‐2048).