Proteins in endplate chondrocytes were lysed and collected, and the protein concentration was determined by the bicinchoninic acid quantitative method. Denatured protein was separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis at 20 g per pore and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin for 1 hour, and primary antibodies against METTL3 (1:1000; Abcam), IL‐1β (1:100, Abcam), cleaved caspase 3 (1:100, Abcam), cleaved caspase 9 (1:100, Abcam), cleaved poly(ADP‐ribose) polymerase (PARP; 1:500, Abcam), ACAN (1:100, Abcam), COL2A1 (1:5000, Abcam), GAPDH (1:5000, Abcam), PIK3R2 (1:5000, Abcam), pAKT (1:5000, Abcam), AKT (1:500, Abcam) or DGCR8 (1:1000, Abcam) were added. The membrane was then incubated at 4°C overnight. The following day, a secondary antibody (1:5000; Cell Signaling Technology, Danvers, MA) was added and the membrane was incubated again for 1 hour at room temperature on a shaker. After washing the membrane three times with Tris‐buffered saline containing Tween 20, images were acquired with a gel imaging system.
Cleaved poly adp ribose polymerase parp
Manufactured by Abcam
Sourced in United States
Cleaved poly(ADP‐ribose) polymerase (PARP) is a protein fragment that is generated during apoptosis, a process of programmed cell death. PARP is involved in DNA repair, and its cleavage is a hallmark of apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Abcam (2 mentions)
Gapdh, by Abcam (2 mentions)
Pik3r2, by Abcam (1 mentions)
Mettl3, by Abcam (1 mentions)
P akt, by Abcam (1 mentions)
Col2a1, by Abcam (1 mentions)
Dgcr8, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
Cleaved caspase 9, by Abcam (1 mentions)
Bicinchoninic acid method, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Merck Group (1 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
Actin, by Santa Cruz Biotechnology (1 mentions)
Hrp labeled anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
3 protocols using cleaved poly adp ribose polymerase parp
1
Protein Expression Analysis in Chondrocytes
2
Western Blot Analysis of Apoptosis Markers
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Total protein was extracted from cells using an RIPA lysis buffer kit (Boster Biological Technology). Protein concentrations were quantified using the bicinchoninic acid method (Thermo Fisher Scientific, Inc.). Following protein separation via 10% SDS-PAGE (40 µg/lane), proteins were transferred onto PVDF membranes (Beyotime Institute of Biotechnology). Following blocking with 5% skimmed milk for 2 h at room temperature, the membranes were then incubated overnight at 4˚C with primary antibodies targeted against: VASH1 (cat. no. ab199732; 1:1,000; Abcam), Bcl-2 (cat. no. ab32124; 1:1,000; Abcam), Bax (cat. no. ab32503; 1:1,000; Abcam), cleaved caspase-3 (cat. no. ab32042; 1:500; Abcam), caspase-3 (cat. no. ab32351; 1:500; Abcam), cleaved poly (ADP-ribose) polymerase (PARP; cat. no. ab32064; 1:1,000; Abcam), PARP (cat. no. ab191217; 1:1,000; Abcam) and GAPDH (cat. no. ab9485; 1:2,500; Abcam). After washing three times with TBST (10% Tween-20), the membranes were then incubated with the HRP-conjugated goat anti-rabbit secondary antibody (cat. no. ab6721; 1:2,000; Abcam) for 1 h at room temperature. The signals were developed using an enhanced chemiluminescence kit (EMD Millipore). Densitometric analysis was performed using ImageJ (version 1.48v; National Institutes of Health).
3
Apoptosis Signaling Pathway Analysis
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Lung tissues from mice and NCI-H460 human lung cells were homogenized with a Dounce homogenizer in lysis buffer at 4 °C. The homogenates were centrifuged at 15,000× g for 20 min at 4 °C, and the supernatants were transferred to different tubes, followed by measuring the protein concentrations. Samples (30 μg of protein) were subjected to 8–12% polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred onto nitrocellulose membranes, which were incubated with primary antibodies (Cleaved poly (ADP-ribose) polymerase (PARP) (Abcam, Cambridge, MA, USA); Cleaved caspase 3, p53 upregulated modulator of apoptosis (PUMA) (Cell Signaling Technology, Danvers, MA, USA); Actin, BAX, Cytochrome c, p53 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Prx-SO3 (AbFrontier, Seoul, Korea)), followed by incubation with HRP-labeled anti-rabbit IgG (ThermoScientific, Waltham, MA, USA). Immunoreactive bands were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Amersham, Buckinghamshire, UK), and band intensities were quantified with ImageQuant 5.2 software (Molecular Dynamics Ltd., Cambridge, CB2 8PQ, UK).
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