Cleaved caspas 8

The isolation of CuC from leaves and fruits of cucumber has been described previously (Qing et al., 2014 (link)). In short, green leaves or fruits were soaked in 95% alcohol at room temperature, then evaporated and separated on silica gel column eluted with chloroform and methanol, and further purified by semi-preparative high-performance liquid chromatography (HPLC) system. The purity of CuC was detected by liquid chromatography–mass spectrometry (LC-MS) analysis (Supplementary Figure 2). The compounds were prepared as a 20 mM stock solution in DMSO. The stock solutions were stored in aliquots at −20°C and diluted with culture medium.
The following antibodies were used in this study: cyclin A, cyclin D1, p21, p27, and p53 (DO-1) were bought from Santa Cruz; Apoptosis Antibody Sampler Kit (9915T), bcl-2, caspas-8, cleaved caspas-8, Akt, p-Akt, β-actin, and GAPDH were bought from Cell Signaling Technology.

The total proteins of 293T cells were extracted with lysis buffer [20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, supplemented with 0.1% PMSF (Beyotime, China)]. The lysates were shaken on ice for 15 min, and the protein concentration was ascertained by BCA assay. The cell lysates were mixed with 2× SDS sample loading buffer and subjected to 10% or 12% (dependent on predicted protein molecular weight) SDS polyacrylamide gel electrophoresis analysis. The proteins were transferred to a PVDF membrane. After blocking with 5% skim milk in TBS-T (0.12 M Tris-base, 1.5 M NaCl, 0.1% Tween-20), the membrane was incubated with primary antibodies, targeting Cleaved-caspase-3, Cleaved-caspase-9, Cleaved-caspas-8, Cleaved-PARP, Bax, Bak, Bid, Smac/Diablo, cIAP-2, Bcl-2, and Rb (all purchased from Cell Signaling Technologies, Danvers, MA, United States), at 4°C overnight. The blots were washed three times in TBST (20 mM pH 7.4 Tris-HCl, 150 mM NaCl, 0.05% Tween-20) and incubated with secondary goat anti-rabbit/mouse horseradish peroxidase-conjugated IgG. The membranes were washed thrice with TBST and the protein was visualized using an ECL chemiluminescent substrate according to the manufacturer’s instructions (Pierce-Thermo Fisher Scientific, Waltham, MA, United States).