Downstream analysis of these six most potent extracts was done using various biological assays, such as Caspase-3/7 activity, mitochondrial membrane potential (MMP), PARP-1 cleavage and expression of γH2Ax, Caspase-8 and -9 using western blotting.
Following detailed analysis using various PKC δ mutants, we determined that PKC δ activation was mediated by caspase-dependent proteolysis.
Cells were treated with ABL-N, and the caspase-2, caspase-3/7, caspase-6, caspase-8, and caspase-9 activities in the cleared lysates were measured by using Caspase-Glo 2, Caspase-Glo 3/7, Caspase-Glo 6, Caspase-Glo 8, and Caspase-Glo 9 assays (Promega) according to the manufacturer's protocols.
Caspase-1 enzymatic activity was measured according to the manufacturer's specification by using a caspase assay kit.
The activity of effector caspases was measured by using the homogeneous caspase assay kit (Roche, Mannheim, Germany).
We have also investigated this phenomenon by measuring the activity of caspases by using a colorimetric caspases kit that measures the activity of caspases-3 in the samples.
An active caspase 3 level was assayed by using caspase 3 assay kit (BD Pharmingen) according to the manufacturer's protocol using spectrofluorimeter (Varian).
At 24 h after incubation, cells were stimulated and at 3 h (for Caspase-8) or 4.5 h (for Caspase-3/7) after stimulation, Caspase-3/7 or -8 activity was measured by using Caspase Glo-3/7 or -8 reagents (Promega).
Caspase-8 and caspase-9 maturation were also evaluated through fluorescence microscopy staining by using Carboxyfluorescein FLICA Apoptosis Detection kit Caspase assay (Immunochemistry Technologies, LLC, Bloomington, MN USA).
Caspase 3 activity was assayed in Huh7 cells by using caspase 3 colorimetric kit (Biovision USA).
