All cell extracts were obtained by lysing cells in ice-cold lysis buffer. The undissolved matter was removed by high-speed centrifugation, and the protein percentage of the supernatant was measured by a BCA protein test kit. Each group of samples was loaded on a 10% SDS-PAGE gel, and the bands were transferred to a PVDF membrane. After being blocked with the sealing solution, the membranes were immunolabeled overnight with the following primary antibodies: HIF-1 (1:1000, Cell Signaling Technology, USA), p53 (1:1000, Cell Signaling Technology, USA), P-gp (1:1000, Cell Signaling Technology, USA), Bax (1:1000, Abcam, USA), F-Caspase3 (1:1000, Cell Signaling Technology, USA), C-Caspase3 (1:1000, Cell Signaling Technology, USA), F-Casase9 (1:1000, Cell Signaling Technology, USA), C-Caspase9 (1:1000, Cell Signaling Technology, USA), F-PARP (1:1000, Abcam, USA), C-PARP (1:1000, Abcam, USA), and β-actin antibodies (1:1000, Santa Cruz, USA). After washing, the protein bands on the PVDF membrane were incubated with suitable auxiliary antibodies (1:2000, ICN Pharmaceuticals), while an ECL kit was used for color development. The bands were analyzed after exposure to a ChemiDoc XRS+ gel imager (Bio-Rad, USA), and the protein content was expressed as the relative value of the corresponding internal reference band.
C parp
Manufactured by Abcam
Sourced in United States
C-PARP is a protein that is cleaved during apoptosis (programmed cell death). It is a commonly used marker for the detection of apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Bcl2 associated x (bax), by Abcam (3 mentions)
C caspase 3, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
F caspase3, by Cell Signaling Technology (1 mentions)
C caspase9, by Cell Signaling Technology (1 mentions)
Chemidoc xrs gel imager, by Bio-Rad (1 mentions)
Hif 1, by Cell Signaling Technology (1 mentions)
β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Abcam (1 mentions)
Lc3 2, by Abcam (1 mentions)
Lc3 1, by Abcam (1 mentions)
Cdk1 y15, by Abcam (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Cdc25c, by Abcam (1 mentions)
Ripa lysis solution, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Cyclinb, by Abcam (1 mentions)
C caspase 3, by Abcam (1 mentions)
7 protocols using c parp
1
Protein Expression Analysis in Cellular Extracts
2
Protein Expression Analysis in Lung Cell Lines
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In order to create protein lysates from MRC-5, A549, Calu-3, H1975, and H2228, protease and phosphatase inhibitors were added to radioimmunoprecipitation assay (RIPA) lysis solution (Thermo Fisher Scientific). The bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) was used to measure the amounts of proteins. After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for electrophoresis, polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA) were transferred. Immunoblotting was carried out using primary antibodies against CDC25C, Bax, Bcl-2, LC3 I, LC3 II, p62, cyclin B, CDK1-Y15, CDK1, c-PARP, and GAPDH (all at 1:1,000; Abcam, Cambridge, MA, USA). Post-washing thrice with tris-buffered saline with Tween 20 (TBST), membranes were processed for band visualization using an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific). Using the ChemiDoc system from Bio-Rad (Hercules, CA, USA), the intensities of the protein bands were recorded and subsequently analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Western Blot Analysis of Ox-LDL Induced Proteins
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Briefly, proteins were obtained from THP1 cells treated with ox-LDL or transfected with the construct used in this research using RIPA buffer. Then, the same amount of protein was subjected to 10% dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto a polyvinyl difluoride membrane (PVDF), and blocked by phosphate buffered solution (PBST) containing 5% non-fat skimmed milk. Next, the membranes were incubated with primary antibodies against CDKN2A, B-cell lymphoma 2 (Bcl-2), BCL2 associated X (Bax), cleaved caspase3 (C-caspase3), cleaved poly ADP-ribose polymerase (C-PARP), or GAPDH (1 : 1000; Abcam, Cambridge, MA, USA), and were then incubated with relative secondary antibodies (1 : 400; Abcam). Finally, a chemiluminescence reagent (Beyotime, Haimen, China) was applied to visualize the protein bands.
4
Evaluating Apoptosis Markers in Lung Tissue
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The lung tissue homogenate of mice in each group was lysed by RIPA lysate at 4°C for 30 min and centrifuged at 12000 rpm for 20 min at 4°C, and the total protein solution was obtained by collecting supernatant. The protein concentration of the samples was determined by the BCA method, and the 20-μg protein samples were separated by SDS-PAGE electrophoresis and transferred to the PVDF membranes. They were then incubated with primary antibodies including Bcl-2 (ab182858; dilution, 1:2000; Abcam), Bax (ab182733; dilution, 1:2000; Abcam), c-caspase 3 (#9661; dilution, 1:1000; Cell Signaling Technology), c-PARP (ab32064; dilution, 1:1000; Abcam), caspase 3 (#9662; dilution, 1:1000; Cell Signaling Technology), PARP (ab191217; dilution, 1:1000; Abcam), SphK1 (cat. no. PA5-14068; dilution, 1:2000; Thermo Fisher Scientific), S1PR1 (cat. no. A70547-050; dilution, 1:1000; EpiGentek), and GAPDH (ab9485; dilution, 1:2500; Abcam) and incubated with a secondary antibody conjugated with horseradish peroxidase. Electrochemiluminescence (ECL) was used to present the bands of the targeted proteins in lung tissue.
5
Comprehensive Immunohistochemical Analysis of Cellular Markers
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For immunohistochemical analysis, formalin-fixed paraffin-embedded (FFPE) tissue sections were deparaffinized and rehydrated using an ethanol series. The antigen was retrieved with 0.01 M citrate buffer (pH 6.0) by heating the sample in a microwave for 10 min. The tissue sections were then placed in 3% hydrogen peroxide for 5 min to inactivate endogenous peroxidases. Primary antibodies against c-PARP (1:100; Abcam), cyclin D1 (1:50; Abcam), and c-Myc (1:300; Novus Biologicals, Centennial, CO, USA) were used for IHC staining. Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Vector Laboratories. c-PARP, cyclin D1, and c-Myc levels were observed using a Pannoramic digital slide scanner system (3D HISTECH; Budapest, Hungary). Antibody validation was performed by serially diluting the antibodies in tissue sections on slides. The positive control experiment was performed using slides with tissue sections from cell-derived xenograft models, and the negative control experiment was performed by replacing the primary antibody with PBS.
6
Multiparametric Immunofluorescence Analysis of FFPE Tissue
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Haematoxylin and eosin (H&E) staining of FFPE sections was conducted using standard approaches by Core Biotechnology Services, University of Leicester. mIF was performed as previously described in detail [37 (link), 38 ] and antibodies were paired with Opal fluorophores (Akoya Biosciences, UK) as follows: CD20Cy (DAKO, Clone L26, 1:200) (Opal 620); CD3 (DAKO, A0452, 1:200) (Opal 520); cPARP (Abcam [E51] 1:2000) (Opal 480); Ki67 (DAKO MIB1, 1:1000) (Opal 570); Cytokeratin (DAKO, Clone AE1/AE3, 1:400) (Opal 690). Whole slides were scanned using a Vectra Polaris (Akoya Biosciences, UK) in MOTIF imaging mode using a 20× objective lens. Images were then analysed using Inform (V2.4), where tissue was segmented into tumour, stroma, necrotic areas, and background/glass. Subsequently, individual stains were assessed, and a merged view obtained (all stains together with DAPI). Phenotyping for Ki67+ve (proliferation), cPARP+ve (apoptosis), CD3+ve (T cell), CD20+ve (B cell), and DAPI+ve (negative) cells was carried out. All six to nine pieces of tissue were treated as a whole for each well/concentration i.e. additive results. A mean was then derived from the six patient samples to provide a combined result.
7
Immunofluorescence Analysis of Cell Apoptosis
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The HT29 and SW620 cells were seeded on the coverslips in a12-well plate (NEST, USA) for 48 h. After washing with cold PBS, the cells were xed in 4% paraformaldehyde for 20 min. Subsequently, the coverslips were permeabilized with 0.5% Triton X-100 for 10 min and blocked with 5% normal goat serum for 1 h at room temperature. Then, the cells were incubated at 4°C with primary antibodies against C-PARP (Abcam, 1:200, USA) overnight, and followed by incubation with fuorophore-conjugated respective secondary antibody (1:200, Invitrogen, USA) for 2 h. The nuclei of cells were visualized by staining with DAPI solution (Sigma, 1:500, USA) for 15 min in the dark. Finally, the slides were observed under a uorescence microscope (Olympus, Japan), and the integrated uorescence density was measured by the ImageJ software.
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