Anti cleaved poly adp ribose polymerase parp

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.

Total protein was extracted from the fibroblasts when they reached 60–80% confluence and protein concentrations were measured using a Bicinchoninic Protein Assay kit (Thermo Fisher Scientific, Inc.). A total of 10 µg protein was separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Following the blocking of non-specific binding with 5% non-fat milk dissolved in TBS + 0.05% Tween-20 at room temperature for 2 h, the membranes were incubated with the following antibodies: Anti-cleaved poly [ADP-ribose] polymerase [PARP; 1:1,000; catalog no. 5625; Cell Signaling Technology, Inc. (CST), Danvers, MA, USA], anti-GAPDH (1:1,000; catalog no. 8884; CST), anti-B cell lymphoma 2 (Bcl-2; 1:1,000; catalog no. 4223; CST), anti-Bax (1:1,000; catalog no. 5023; CST) and anti-mothers against decapentaplegic homolog 3 (Smad3; 1:1,000; catalog no. ab40854; Abcam, Cambridge, UK) at 4°C overnight. Following washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (1:1,000; catalog no. 7074; CST) and the protein expressions were visualized using the Enhanced Chemiluminescence System (EMD Millipore, Billerica, MA, USA). The bands were quantified by densitometry using Image J 1.46r software (National Institutes of Health, Bethesda, MD, USA). All reactions were performed in triplicate.

Immunoblot analyses were carried out as previously described.
¹⁸ (link) The membranes were incubated with antibodies against anti‐DYRK2 (Sigma‐Aldrich), anti‐p53 (Santa Cruz Biotechnology), anti‐phospho‐p53‐Ser46 (Bio Academia and Cell Signaling Technology), anti‐cleaved poly (ADP‐ribose) polymerase (PARP) (Cell Signaling Technology), anti‐cleaved caspase‐3 (Cell Signaling Technology) and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology). Then membranes were incubated with peroxidase‐conjugated anti‐rabbit IgG (Cell Signaling Technology) or anti‐mouse IgG (Cell Signaling Technology). The dilution ratios were all 1:1000.