The HEL 92.1.7 and SET-2 cells (1 × 106) treated with CR-LAAO (0.03–0.15 μg/mL and 1.5–3.0 μg/mL, respectively) were suspended in 200 μL of the Western blotting sample buffer supplemented with 5% mercaptoethanol, 4% sodium dodecyl sulfate (SDS), 20% glycerol, and 100 mM Tris–HCl at pH 6.0. The samples were analyzed by electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes.
The membrane was sequentially labeled overnight with anti-caspase-3 polyclonal rabbit antibody (code 9662, Cell Signaling Technology), anti-tubulin (code T3320, Sigma–Aldrich), anti-caspase-9 polyclonal rabbit antibody (code 9502, Cell Signaling Technology), anti-caspase-8 mouse monoclonal antibody (code 9746, Cell Signaling Technology), and anti-cleaved Poly(ADP-ribose) polymerase (PARP – code 9541, Cell Signaling Technology). The antibodies had been diluted in a blocking solution containing 5% skim milk and 0.01% sodium azide, according to the manufacturers’ instructions.
After labeling with each primary antibody, the membrane was incubated with the peroxidase-labeled anti-mouse or anti-rabbit secondary antibody for 1 h at room temperature. The labeled proteins were detected by chemiluminescence (Amersham ECL Plus, GE Healthcare Life Science, Pittsburgh, BREAK). Then, the membrane was washed with the stripping buffer before being relabeled with the next primary antibody.
The membrane was sequentially labeled overnight with anti-caspase-3 polyclonal rabbit antibody (code 9662, Cell Signaling Technology), anti-tubulin (code T3320, Sigma–Aldrich), anti-caspase-9 polyclonal rabbit antibody (code 9502, Cell Signaling Technology), anti-caspase-8 mouse monoclonal antibody (code 9746, Cell Signaling Technology), and anti-cleaved Poly(ADP-ribose) polymerase (PARP – code 9541, Cell Signaling Technology). The antibodies had been diluted in a blocking solution containing 5% skim milk and 0.01% sodium azide, according to the manufacturers’ instructions.
After labeling with each primary antibody, the membrane was incubated with the peroxidase-labeled anti-mouse or anti-rabbit secondary antibody for 1 h at room temperature. The labeled proteins were detected by chemiluminescence (Amersham ECL Plus, GE Healthcare Life Science, Pittsburgh, BREAK). Then, the membrane was washed with the stripping buffer before being relabeled with the next primary antibody.