Cells were plated in six-well plates at a concentration of 5 × 105 cells/well in an antibiotic-free medium one day before transfection. We harvested cells at 48, 96, and 120 h following transfection, using trypsin-EDTA (Mediatech) and dissolved in NP40 lysis buffer (0.5% NP40, 250 mM NaCl, 50 mM Hepes, 5 mM EDTA, 0.5 mM egtazic acid) freshly supplemented with a complete protease inhibitor and phosphatase inhibitor cocktails 1 and 2 (Roche). Proteins were purified and the levels of PARP protein quantified with the rabbit polyclonal anti-PARP1 antibody (Cell Signaling Technology) using standard procedures for Western blotting. Normalization was performed with mouse monoclonal anti-ACTB antibody (Cell Signaling Technology). For further confirmation of apoptosis, Colo320, SW620, SW480, RKO, and HCT116 cell lines were analyzed using the Caspase-3/7, 8, and 9 assays according to the manufacturer’s protocol (Promega, Madison, WI, USA). To dissect the detailed pathway of apoptosis, we used antibodies specific to XIAP (Cell Signaling Technology) and c-IAP1 (Cell Signaling Technology).
Anti parp1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-PARP1 antibody is a laboratory reagent used to detect and study the expression of the PARP1 protein. PARP1 is an enzyme involved in various cellular processes, including DNA repair and programmed cell death. This antibody can be used in techniques such as Western blotting, immunoprecipitation, and immunohistochemistry to identify and quantify PARP1 in biological samples.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Anti β actin, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Ciap1, by Cell Signaling Technology (1 mentions)
Anti parp2 antibody, by Active Motif (1 mentions)
Anti par antibody, by R&D Systems (1 mentions)
Anti α tubulin antibody, by Merck Group (1 mentions)
Hl 60, by Fujifilm (1 mentions)
P brca1, by Santa Cruz Biotechnology (1 mentions)
Hl 60, by American Type Culture Collection (1 mentions)
I bet151, by Selleck Chemicals (1 mentions)
5 azacytidine, by Fujifilm (1 mentions)
Horseradish peroxidase labeled anti rabbit igg antibody, by GE Healthcare (1 mentions)
β actin, by Abcam (1 mentions)
Anti brca1 antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Anti β tubulin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
11 protocols using anti parp1 antibody
1
Apoptosis Induction and PARP1 Quantification
2
Western Blot Antibody Validation
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Primary antibodies used in this study for western blot include: anti-PARP1 antibody (Cell Signaling Technology, 9542, 1:2,000), anti-PARP2 antibody (Active Motif, 39044, 1:2,000), anti-α-Tubulin antibody (Sigma, CP06, 1:1,000), anti-pan-ADP-ribose binding reagent (Sigma, MABE1016, 1:1,000), anti-β-actin (Sigma, A5441, 1:10,000), and anti-PAR antibody (R&D, 4335-MC-100, 1:1,000).
3
Establishing AZA-Resistant Cell Lines
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U937 and HL-60 cells were purchased from ATCC (Manassas, VA, United States). AZA-resistant cell lines (R-U937 and R-HL-60) were originally created in our laboratory from U937 and HL-60 cells, respectively (Imanishi et al., 2014 (link)).
5-Azacytidine was purchased from Wako Pure Chemical Industries (Osaka, Japan) and I-BET151 from Selleck Chemicals (Houston, TX, United States). The antibodies specific for HP1α, HP1β, HP1γ, and β-actin were purchased from Abcam (Cambridge, United Kingdom). The antibodies specific for BRCA1 phosphorylated at Ser 1423 (p-BRCA1), total BRCA1 and total ATM were from Santa Cruz Biotechnology (Dallas, TX, United States) and anti ATM phosphorylated at Ser 1981 (p-ATM) antibody was from R&D Systems (Minneapolis, MN, United States). Anti PARP1 antibody was from Cell Signaling Technologies (Danvers, MA, United States). The secondary antibodies, namely horseradish peroxidase–labeled anti-mouse IgG antibody and horseradish peroxidase–labeled anti-rabbit IgG antibody, were purchased from GE Healthcare (Buckinghamshire, United Kingdom).
5-Azacytidine was purchased from Wako Pure Chemical Industries (Osaka, Japan) and I-BET151 from Selleck Chemicals (Houston, TX, United States). The antibodies specific for HP1α, HP1β, HP1γ, and β-actin were purchased from Abcam (Cambridge, United Kingdom). The antibodies specific for BRCA1 phosphorylated at Ser 1423 (p-BRCA1), total BRCA1 and total ATM were from Santa Cruz Biotechnology (Dallas, TX, United States) and anti ATM phosphorylated at Ser 1981 (p-ATM) antibody was from R&D Systems (Minneapolis, MN, United States). Anti PARP1 antibody was from Cell Signaling Technologies (Danvers, MA, United States). The secondary antibodies, namely horseradish peroxidase–labeled anti-mouse IgG antibody and horseradish peroxidase–labeled anti-rabbit IgG antibody, were purchased from GE Healthcare (Buckinghamshire, United Kingdom).
4
Protein Expression Analysis in SCLC Cells
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The total protein from SCLC cells was extracted using RIPA lysis buffer. The protein lysates were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Millipore, USA). The membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies. The signals were detected using an ECL system (Thermo Fisher Scientific, USA). The intensity of the protein fragments was quantified with Image Lab software (Version 5.2.1 build 11, Bio-Rad). Anti-PARP1 antibody (Cell Signaling Technology, USA), anti-BRCA1 antibody (Santa Cruz, USA), anti-Rad51 antibody (Abcam, USA), anti-GAPDH and anti-β-tubulin (Cell Signaling Technology, USA) were used as the primary antibodies for the detection of specific proteins.
5
PARP1 Knockout in U2OS Cells
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U2OS or 293 T cells were maintained in DMEM medium with 10% fetal bovine serum and cultivated at 37 °C in 5% CO2 (v/v). U2OS cells were transfected with PX459 vector containing PARP1-sgRNA for PARP1 knockout. Transfected cells were plated at low density in 1.5 mg/ml puromycin (Invitrogen). Single colonies were propagated, and individual clones were assessed by western blotting. Loss of PARP1 in U2OS cells was validated by anti-PARP1 antibody which was purchased from Cell Signaling Technology (Cat# 9542).
Cells were lysed with NETN buffer (0.5% NP40, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM EDTA) with Roche Protease Inhibitor Cocktail. Immunoprecipitation and Western blotting were performed following standard protocol as described previously20 (link). Rabbit anti-CTCF antibody was purchased from Cell Signaling. Monoclonal anti-PAR antibody was purchased from Trevigen. Mouse monoclonal anti-GFP antibody was purchased from Cell Signaling.
Cells were lysed with NETN buffer (0.5% NP40, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM EDTA) with Roche Protease Inhibitor Cocktail. Immunoprecipitation and Western blotting were performed following standard protocol as described previously20 (link). Rabbit anti-CTCF antibody was purchased from Cell Signaling. Monoclonal anti-PAR antibody was purchased from Trevigen. Mouse monoclonal anti-GFP antibody was purchased from Cell Signaling.
6
Protein Expression Analysis in Tumor Tissues
7
Triptolide Modulates Oxidative Stress and Apoptosis
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Triptolide was purchased from Chengdu Biopurify Phytochemicals Ltd. Cholesterol (Chol), distearoyl phosphatidylglycerole (DSPG) and egg yolk lecithin (PC-98T) were sourced from AVT (Shanghai) Pharmaceutical Tech Co., Ltd., Shanghai, China. GSH and GSSG assay kit, Annexin V-FITC/Propidium iodide (PI) Apoptosis Detection Kit, Cell Cycle and Apoptosis Analysis Kit, Bicinchoninic Acid (BCA) Protein Assay Kit and goat anti-rabbit IgG/HRP antibody were obtained from Beyotime Institute of Biotechnology, Nanjing, China. Reactive Oxygen Species Assay Kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. RPMI 1640 medium, trypsin, and fetal bovine serum (FBS) were provided by Gibco BRL, USA. Anti-caspase-3 antibody and anti-PARP-1 antibody were soured from Cell Signaling Technology, USA. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) rabbit monoclonal antibody was purchased from WuXi AppTec, Shanghai, China.
8
Modulation of Apoptosis via Bcl-xL
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300,000 HeLa cells were plated in six-well plates. 24 h after seeding, cells were transiently transfected with 1 µg of empty pCMV24 vector, pCMV24-3xFLAG-Bcl-xL or pCMV24-3xFLAG-Bcl-xLK87D constructs. X-tremeGene HP DNA (Roche) was used as a transfection reagent according to the manufacturer’s instructions. 48 h after transfection, HeLa cells were treated with 0.5 µM staurosporine (Sigma-Aldrich) for 6 h or with 25 µM venetoclax (Cayman Chemical/Sanbio, Uden, Netherlands). DMSO (Invitrogen) was used as vehicle. Cells were then harvested and lysed as previously described [11 (link)]. Apoptosis was monitored via western-blotting with an anti-PARP1 antibody (Cell Signaling Technology, #9532) that detected both cleaved and uncleaved PARP. PARP cleavage was calculated as the ratio of cleaved PARP over total PARP. Quantifications were performed using the ImageJ software (National Institutes of Health, USA). We also used anti-Bcl-xL (Cell Signaling Technology; #2764), anti-β-actin (Sigma-Aldrich; A5441) and anti-vinculin (Sigma-Aldrich; V9131) antibodies.
9
Immunoblot Analysis of DNA Damage Markers
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Cells treated with CCC‐018‐TPP were lysed in radioimmunoprecipitation assay (RIPA) buffer containing Complete Protease Inhibitor Cocktail (Merck) and PhosSTOP (Merck Millipore). The lysates were centrifuged at 10 000× g for 10 minutes at 4°C, and the supernatant was used for immunoblot analysis. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) under reducing conditions and transferred to an Immobilon‐P transfer membrane (Merck Millipore). The membrane was blocked with BLOCK ACE (DS Pharma Biomedical Co., Ltd.) or 5% BSA in TBS‐T (150 mmol/L NaCl, 20 mmol/L Tris‐HCl, pH 7.5, 0.1% Tween 20). The primary antibodies used were a rabbit polyclonal anti‐PARP‐1 antibody (Cell Signaling Technology), a mouse monoclonal anti‐γ‐H2AX antibody (Cell Signaling Technology), and a mouse monoclonal anti‐β‐actin antibody (Santa Cruz Biotechnology). All primary antibodies were used at 1:1000 dilutions. The membrane was washed extensively with TBS‐T and then incubated with the appropriate horse radish peroxidase (HRP)‐conjugated secondary antibody (1:3000 dilution). ECL Plus Western Blotting Detection Reagent (Amersham Biosciences) was used for immunodetection. The membranes were scanned with a Luminoimaging Analyzer LAS4000 (GE Healthcare), and the bands were quantified via ImageJ software.
10
Reagents and Cell Lines for Cancer Research
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Fetal bovine serum, trypsin, and Hank’s balanced salt solution were purchased from Invitrogen (Carlsbad, CA). RPMI 1640 medium was obtained from Welgene (Gyeongsan-si, Republic of Korea), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from EMD Millipore (Billerica, MA), and M2 anti-FLAG antibody was obtained from Sigma-Aldrich (Saint Louis, MO). Basic fibroblast growth factor and epidermal growth factor were purchased from R&D Systems (Minneapolis, MN). Cell culture plates with ultra-low attachment surfaces were purchased from Corning Life Sciences (Tewksbury, MA). Neurobasal medium, RPMI 1640, FBS, B-27 Supplement, penicillin, streptomycin, and Accutase cell detachment solution were purchased from Life Technologies (Grand Island, NY). The epithelial human ovarian cancer cell line A2780 was purchased from the American Type Culture Collection (Manassas, VA). Cell cycle regulation antibody sampler kit and anti-PARP-1 antibody were purchased from Cell Signaling Technology, Inc. (Danvers, MA).
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