Cleaved anti human parp

Western blotting was run as previously described with certain modifications adjusted to the molecular weight of certain protein. Anti-human cIAP1, anti-human Caspase-8, cleaved anti-human Caspase-8, cleaved anti-human Caspase-3, cleaved anti-human PARP, anti-human c-FLIP, and anti-human NF-κB (p65) were all purchased from Cell Signaling Technology (Cambridge, U.K.). β-actin was used as loading control in the early stage while GAPDH was a better candidate whenever apoptosis may happen. Histone-3 was used as loading control for nuclear fractions. Mild lysate NP-40 (Beyotime, China) other than RIPA was applied to avoid unexpected apoptosis [19 (link)].
The cells were pre-treated with z-VAD to prevent further reaction and incubated with stimulation of TNF-α (40 ng/ml)/cycloheximide (5 mg/ml) for 6 h [20 (link)]. An amount of 1:50 anti-human Caspase-8 or the same amount of IgG antibody together with Protein A/G Plus-Agarose (Santa Cruz Biotechnology) were added within the reaction system to incubate the complexes overnight. The mixtures were washed for three times with NP-40 by centrifuging, resuspended with 1× loading buffer, and subscribed to SDS/PAGE blotting for interested proteins. Each experiment was repeated in triplicate.

Cells transfected with lentiviral vectors were lysed with lysis buffer at 4°C for 30 min. The concentration of cells was measured by the BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were then sealed with non-fat milk in tris-buffered saline with Tween for 3 h. The appropriate diluted primary antibodies, including anti-human LHPP (1:500; Invitrogen Carlsbad, CA, U.S.A.), anti-human cIAP1 (1:1000; Cell Signaling Technology, Danvers, MA, U.S.A.), cleaved anti-human Caspase-3 (1:500; Abcam, Cambridge, U.K.), cleaved anti-human PARP (1:1000; Cell Signaling Technology), anti-human p53 (1:1000; Cell Signaling Technology), anti-human AKT-pS473 (1:3000; Proteintech, Chicago, IL, U.S.A.), anti-human AKT (1:1000; Cell Signaling Technology), anti-human PTEN (1:1000; Cell Signaling Technology), and anti-human GAPDH (1:2000; Servicebio, Wuhan, China) were then incubated with the membranes overnight at 4°C. The second antibody was diluted with TBST and incubated with the membranes for 1 h at room temperature. Immunoreactivity was determined using a chemiluminescence western blot immunodetection kit (Invitrogen) according to the manufacturer’s instructions. The amounts of each protein were semi-quantified, and GAPDH was regarded as an internal control.