Tissues were homogenized in NP-40 lysis buffer (50 nM Tris-HCl pH 8.0, 100 mM Nacl, 0.5% NP-40) supplemented with 1X complete protease inhibitor (Roche, Indianapolis, IN, USA). 100 μl of protein lysate was resolved on 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. IP was performed as described in (25 ). After transfer on nitrocellulose membranes, blots were probed with p53 (CM5, 1:1000, Novacastra, IL, USA), Mdm2 (2A10, 1:500, Calbiochem, San Diego, CA, USA), Mdm4 (1:500; MX82, Sigma), Actin (A5441, 1:5000, Sigma, St Louis, MO, USA), Cleaved PARP-1 (1051–1, 1:1000, Epitomics/Abcam, Cambridge, UK), p21 (556431, 1:1000, BD Pharmingen, USA) and Vinculin (V9131, 1:5000, Sigma, St Louis, MO, USA) antibodies. Data were analyzed using ImageJ software (National Institutes of Health, Bethesda).
Cleaved parp1
Manufactured by Abcam
Sourced in United States, United Kingdom
Cleaved PARP1 is a protein fragment that is generated as a result of the cleavage of the Poly(ADP-ribose) polymerase 1 (PARP1) enzyme. PARP1 is involved in the cellular response to DNA damage, and its cleavage is often used as a marker of apoptosis or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
Bcl 2, by Abcam (5 mentions)
Caspase 3, by Abcam (5 mentions)
Cleaved caspase 3, by Abcam (4 mentions)
β actin, by Abcam (4 mentions)
Parp1, by Abcam (4 mentions)
Bcl2 associated x (bax), by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
P iκbα, by Cell Signaling Technology (2 mentions)
Nf κb p65, by Cell Signaling Technology (2 mentions)
Ps15 p53, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
P mtor, by Abcam (2 mentions)
P akt, by Abcam (2 mentions)
P pi3k, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
β catenin, by Abcam (2 mentions)
Bcl xl, by Abcam (2 mentions)
Ab32064, by Abcam (2 mentions)
24 protocols using cleaved parp1
1
Western Blot Analysis of p53 Signaling
2
Quantifying Protein Expression Changes
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Equal amounts of soluble protein from cellular lysate or hippocampal homogenate were electrophoretically separated using 10%-15% SDS-PAGE gel and transferred onto a nitrocellulose membrane. After blocking, membranes were incubated with primary antibodies speci c for TNF-α, cleaved caspase-3, BACE1, p-IκB-α, IκB-α, p-NF-κB p65, and NF-κB p65(Cell Signaling Technology); cleaved PARP-1, APP, Iba-1, GFAP,AdipoR1, TLR4, and CD14 (Abcam) at 4°C overnight. The membranes were probed with HRP-conjugated secondary antibodies, and immunoreactive images were exhibited using a chemiluminescent detection system and collected on Bio-Rad Chemidoc Imaging System, and the intensities of the protein bands were determined using Image-Pro Plus 6.0 software.
3
Investigating ZINC20032678 and Inhibitors Effects
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Proteins were extracted from MDA-MB-231 cells treated with 0, 0.1, 1 and 10 μmol/L ZINC20032678 and 1 μmol/L crizotinib (c-Met inhibitor) or NMS-P118 (PARP-1 inhibitor) at 72 h, respectively, and the untreated cells were set as the blank control. Then we further separated by using a 10% polyacrylamide gel. After transferring the protein on a nitrocellulose membrane, the membrane was blocked with a 5% defatted milk solution and probed with recombinant monoclonal antibody against phosphorylated c-Met (1:2000, Abcam, USA), full-length monoclonal PARP-1 (1:1000, Abcam, USA), cleaved PARP-1 (1:1000, Abcam, USA), cleaved caspase-3 (1:500, Abcam, USA), Bcl2 (1:2000, Abcam, USA), Bax (1:2000, Abcam, USA) and β-actin (1:5000, Abcam, USA), and then probed with a secondary antibody using ALP conjugated anti-human IgG (1:5000, Santa cruz, USA), at last, blots were developed using a ECL plus kit (GE, USA).
4
Western Blot Protein Analysis
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The Western blot assay was performed as previously described in detail [19] (link). GAPDH, β-actin (Santa Cruz Biotech, CA, USA), cleaved Caspase-3 (Cell Signaling, Beverly, MA, USA), γ-H2AX, and cleaved PARP-1 (Abcam, Cambridge, UK) antibodies were used. β-actin or GAPDH was used as a loading control. The protein bands were visualized using enhanced chemiluminescence detection reagents (Advansta, Menlo Park, CA, USA) on an LAS system (Las 4000 mini, GE Health Care, USA). Densitometric analysis was performed using ImageJ software.
5
Immunoblotting of Cell Cycle Regulators
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Immunoblotting was carried out as previously described [40 (link)] using the following primary antibodies: CDK7 (Cell Signaling Technology, 2916), CDK1 (Cell Signaling Technology, 9116), CDK2 (Cell Signaling Technology, 2546), CDK4 (Cell Signaling Technology, 12790), CDK9 (Cell Signaling Technology, 2316), β-actin (Abcam, ab6276), GAPDH (Cell Signaling Technology, 2118), P-S5 PolII (Abcam, ab5401), P-S2 PolII (Abcam, ab5095), PolII (Abcam, ab817), P-S780 Rb (Abcam, ab47763), P-S807/811 Rb (Cell Signaling Technology, 8516), Rb (Abcam, ab6075), cyclin H (Abcam, ab54903), MAT1 (Santa Cruz Biotechnology, sc135981), P-S15 p53 (Cell Signaling Technology, 9284), p53 (Santa Cruz Biotechnology, sc-126), P-T161/T160 CDK1/CDK2 (Abcam, ab201008), P-S10 Histone H3 (Abcam, ab139417), AR (MilliporeSigma, 06-680), P-T1457 MED1 (Abcam, ab60950), MED1 (Bethyl Laboratories, A300-793A), cleaved PARP1 (Abcam, ab4830), p21 (Santa Cruz Biotechnology, sc-817).
6
Western Blot Analysis of Protein Expression
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The total protein was extracted from rat intestinal tissues or IEC-6 cells adopting RIPA buffer (Mlbio, China) and then subjected to 10% SDS-PAGE for separation, followed by transferring to PVDF membranes. Thereafter, the membranes were blocked with 5% non-fat milk for 2 h and then immunoblotted with primary antibodies overnight at 4°C and goat anti-rabbit HRP antibody (cat. no. ab205718; 1/2000; Abcam) for 1 h at room temperature. The blots were visualized by the ECL reagent (Mlbio, China) and the gray analysis was implemented with ImageLab4.0 software. Inducible nitric oxide synthase (iNOS; cat. no. ab178945; 1/1000; Abcam), cyclooxygenase-2 (COX2; cat. no. ab179800; 1/1000; Abcam), cleaved caspase 3 (cat. no. #9661; 1/1000; Cell Signaling Technology), caspase 3 (cat. no. ab184787; 1/2000; Abcam), cleaved PARP1 (cat. no. ab32064; 1/1000; Abcam), PARP1 (cat. no. ab191217; 1/1000; Abcam), LC3B (cat. no. ab192890; 1/2000; Abcam), Beclin1 (cat. no. ab207612; 1/2000; Abcam), SIRT3 (cat. no. ab189860; 1/1000; Abcam), p-AMPK (cat. no. ab133448; 1/1000; Abcam), AMPK (cat. no. ab207442; 1/1000; Abcam), p-mTOR (cat. no. #5536; 1/1000; Cell Signaling Technology), mTOR (cat. no. ab134903; 1/10,000; Abcam) and GAPDH (cat. no. ab181602; 1/10,000; Abcam) primary antibodies were utilized in this study.
7
Chidamide and Bortezomib Combination Protocol
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Chidamide was provided by Chipscreen Biosciences (Chipscreen Biosciences Co., Ltd., Shenzhen, China) and dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) at a 100 mM concentration as a stock solution. Bortezomib was purchased from Xian Janssen (Xian Janssen Pharmaceutical Co., Ltd., Xian, China) and dissolved in DMSO at the concentration of 1mM as a reserve solution. The two solutions were kept at −80 °C and diluted to the working concentration in subsequent experiments.
Primary antibodies against B-cell lymphoma 2 (Bcl-2), matrix metallopeptidase 2 (MMP2), E-cadherin and N-cadherin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GADPH), cleaved caspase-3, acetyl-H3, acetyl-H14, AKT, p-AKT, PI3K, p-PI3K, cleaved-PARP1, and Bad were purchased from Abcam (Cambridge, MA, USA). Antibodies against cyclin D1 and cyclin E1 were purchased from Cell Signaling Technology (Danvers, MA, USA).
Primary antibodies against B-cell lymphoma 2 (Bcl-2), matrix metallopeptidase 2 (MMP2), E-cadherin and N-cadherin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GADPH), cleaved caspase-3, acetyl-H3, acetyl-H14, AKT, p-AKT, PI3K, p-PI3K, cleaved-PARP1, and Bad were purchased from Abcam (Cambridge, MA, USA). Antibodies against cyclin D1 and cyclin E1 were purchased from Cell Signaling Technology (Danvers, MA, USA).
8
Ubenimex Induces Apoptosis in Cancer Cells
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Ubenimex were provided by Shenzhen Main Luck Pharmaceuticals, Inc. (Shenzhen, China). LIVE/DEADTM Cell Imaging Kit and Total Reactive Oxygen Species (ROS) Assay Kit were purchased from Thermo Fisher Scientific (USA). Cell Counting Kit-8 was purchased from Dojindo (Japan). Annexin V-FITC/PI kit was purchased from BD Biosciences (USA). NAC, 3-MA and Z-VAD-FMK were purchased from Selleck (USA). Primary antibodies: caspase-3 (1:500; catalog # ab13847), parp1 (1:2000; catalog #ab32138), cleaved parp1 (1:2000; catalog #ab32064) and β-actin (1:5000; catalog # ab8226) were purchased from Abcam (UK). ERK (1:1000; catalog # 4695T) and p-ERK (1:1000; catalog # 4370T) were purchased from Cell Signaling Technology (USA). Caspase-9 (1:1000; catalog # 10380-1-AP) was purchased from Proteintech (USA).
9
Western Blot Analysis of Apoptotic and Cell-Cell Adhesion Markers
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Lysis Buffer and BCA Protein Assay Kit from Thermo Fisher were employed to extract total protein and determine their concentrations, respectively. After separation using 10% SDS-PAGE, protein bands were transferred to the PVDF membrane. After 1 h of blocking with 5% non-fat milk, samples were incubated overnight with primary antibodies against BAX (1:1000, Abcam, Boston, MA, USA), BCL2 (1:1000, Abcam, Boston, MA, USA), Caspase3 (1:1000, Abcam, Boston, MA, USA), cleaved PARP1 (1:1000, Abcam, Boston, MA, USA), E-selectin (1:1000, Abcam, Boston, MA, USA), ICAM1 (1:1000, Abcam, Boston, MA, USA), VCAM1 (1:1000, Abcam, Boston, MA, USA), P120-catenin (1:1000, Abcam, Boston, MA, USA), β-catenin (1:1000, Abcam, Boston, MA, USA), and E-cadherin (1:1000, Abcam, Boston, MA, USA) at 4° C. Finally, samples were incubated for 1 h with HRP-conjugated secondary antibodies and the protein bands were analyzed.
10
Apoptosis Protein Expression Analysis
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Cells or tumor tissues were lysed and the proteins were extracted and separated by SDS-PAGE. Then the proteins were transferred to PVDF membrane and incubated with antibodies for caspase-3, caspase-8, PARP-1, cleaved PARP-1, IL-24, E1A and GAPDH (Abcam, UK) at 4°C overnight. Membranes were incubated with secondary antibodies for 2 h. Finally, the protein bands were tested by ECL reagents according to the instructions. The gray value of the bands was analyzed with Image-J software.
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