HeLa, HEC-1-A, HEK293, A549, and Beas-2B cells were lysed in RIPA buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, and 1% Triton 100) at 4 °C. Proteins in the resultant lysates were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against α-actinin (ACTN), ATF3, c-Fos, FASN HuR, Nrf2, p21, p53, p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-CoA carboxylase (ACC), p-ACC (phosphorylation at Ser 79), ERK, p-ERK, HIF-1α, LC3B, cleaved poly-ADP-ribose polymerase (cPARP) (Cell Signaling, Danvers, MA, USA), Cyclin D1 (Abcam, Cambridge, UK), HO-1 (Enzo Life Sciences, Farmingdale, NY, USA), and DEC1 (Bethyl Laboratories, Montgomery, TX, USA).
Cleaved poly adp ribose polymerase
Manufactured by Cell Signaling Technology
Sourced in United States
Cleaved poly-ADP ribose polymerase is a laboratory product that detects the cleaved form of poly-ADP ribose polymerase (PARP), a protein involved in DNA repair. This product is used as a marker for apoptosis, or programmed cell death, in various cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (6 mentions)
β actin, by Merck Group (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
α actinin, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Cytiva (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
Cyclin b1, by Cell Signaling Technology (2 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Poly adp ribose polymerase, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Acetyl coa carboxylase, by Cell Signaling Technology (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Abcam (1 mentions)
Survivin, by R&D Systems (1 mentions)
25 protocols using cleaved poly adp ribose polymerase
1
Protein Expression Analysis in Various Cell Lines
2
Evaluating Apoptotic and Cell Cycle Markers
3
Immunoblotting Analysis of Protein Expression
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Equal amounts (50 μg) of protein extracts were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences). Nonspecific sites were blocked by incubation of the membrane in blocking buffer [5% nonfat dry milk in T-TBS (TBS containing 0.05% Tween 20)] overnight. The membranes were incubated with the indicated primary antibodies [matrix metalloproteinase (MMP)-9 (1 : 5000, Abcam), nuclear factor (NF)-κB (1 : 600, Abcam), tumor necrosis factor (TNF)-α (1 : 1000, Cell Signaling), Bax (1 : 1000, Abcam), caspase-3 (Csp-3) (1 : 4000, Abcam), cleaved poly-ADP-ribose polymerase (c-PARP) (1 : 1000, Cell Signaling), and actin (1 : 10000, Chemicon)] for 1 hr at room temperature. Horseradish peroxidase-conjugated anti-rabbit immunoglobulin IgG (1 : 2000, Cell Signaling) was used as the secondary antibody for 1 hr at room temperature. The washing procedure was repeated 8 times within 1 hr, and immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) and exposure to Biomax L film (Kodak). For purposes of quantification, ECL signals were digitized using Labwork software (UVP).
4
Antibody Immunoblotting of Signaling Proteins
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Antibodies against Akt, phosphorylated Akt, PI3K, phosphorylated PDK1, phosphorylated TSC2, phosphorylated GSK3β, phosphorylated p70S6K, phosphorylated 4EBP1, mTOR, PTEN, FOXO1, PCNA, and cleaved poly ADP ribose polymerase (C-PARP) were from Cell Signaling Technology (Danvers, MA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), survivin, CDK4, and pRb were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phosphorylated mTOR and phosphorylated PTEN were from Abcam (Cambridge, UK). Antibody against Cyclin D1 was from Biosource (Camarillo, CA).
5
Western Blot Analysis of Apoptosis Markers
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Equal amounts (10–30 μg) of protein extracts from cells or myocardium were loaded and separated by SDS-PAGE using 8–10% acrylamide gradients. Following electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Piscataway, NJ, USA). Nonspecific proteins were blocked by incubating the membrane in blocking buffer (5% nonfat dry milk in T-TBS containing 0.05% Tween 20) overnight. The membranes were incubated with antibodies against cleaved caspase-3 (c-Csp 3) (1:1000, Cell Signaling, Danvers, MA, USA), cleaved poly (ADP-ribose) polymerase (c-PARP) (1:1000, Cell Signaling), TGF-β (1:500 Cell Signaling), phosphorylated Smad2 (1:1000 Cell Signaling), Bax (1:1000, Abcam, Cambridge, MA, USA), and β-actin (1:10,000, Millipore) for 1 h at room temperature. Signals were detected with HRP-conjugated goat anti-mouse or goat ant-rabbit with ECL (Perkin Elmer, MA, USA).
6
Protein Expression and Immunoblotting
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Cells were lysed at 4 °C in lysis buffer (100 mM Tris-HCl of pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% Triton X-100). Proteins in the lysate were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA), and probed using antibodies against α-actinin (ACTN), cyclin D1, HSP90 α/β, p53(Santa Cruz Biotechnology, CA, USA), caspase 3, cyclin B1, EGFR, p-Histone H3 (phosphorylation at Ser 10, H3P), cleaved poly-ADP-ribose polymerase (cPARP) (Cell Signaling, MA, USA), and DEC1 (Bethyl Laboratory, TX, USA).
7
Protein Expression Analysis in Cancer Cells
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HeLa and A2058 cells were lysed in RIPA buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% Triton 100) at 4 °C. The protein concentration was measured using a DC Protein Assay Kit (Bio-Rad Laboratories, USA). Proteins in the resultant lysate were separated by SDS-PAGE and then electrotransferred to PVDF membranes (Immobilon-P; Millipore, Bedford, MA, USA) using a Bio-Rad Semi-dry Transfer Cell. The blots were then incubated with primary antibodies against α-actinin (ACTN), Ac-H3 (acetylated form of histone H3 at lysine 9/14), fatty acid synthase (FASN), p21, p53, proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, USA), acetyl-CoA carboxylase (ACC), p-ACC (phosphorylation at Ser 79), AMPK, p-AMPK (phosphorylated at Thr 172), cleaved poly-ADP-ribose polymerase (cPARP) (Cell Signaling, USA), and DEC1 (Bethyl Laboratory, USA). Thereafter, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). The immunoreactive proteins were then detected using ECLTM Western Blotting Detection Reagents and Amersham HyperfilmTM ECL (GE Healthcare, USA).
8
Signaling Pathway Evaluation in Cells
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HJC0152 was obtained from Selleck Chemicals LLC (Houston, TX, USA) and dissolved in dimethyl sulfoxide (DMSO) to prepare a 20 µM stock solution. Primary antibodies against STAT3, p-STAT3 (Y705), p-STAT3 (S727), c-Jun N-terminal kinase (JNK), phospho-JNK, p38 MAPK, and p-p38 MAPK were bought from Abcam (Cambridge, UK) and those against p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2), p-p44/42 MAPK (p-ERK1/2), c-Myc, cyclinD1, survivin, Mcl1, and cleaved-poly(ADP-ribose) polymerase (c-PARP) from Cell Signaling Tech (Beverly, MA, USA). Finally, the anti-glyceraldehyde-3-phosphate dehydrogenase antibody and horseradish peroxidase-conjugated secondary antibodies were purchased from Proteintech (Hubei, People’s Republic of China).
9
Sulforaphane-Induced Cellular Responses
10
Antibody-based Protein Analysis Protocol
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Antibodies that recognize phosphorylated or total proteins, including AKT, p-AKT (S473), S6, p-S6 (240/244), and cleaved poly ADP-ribose polymerase (c-PARP), were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against β-Actin and CD31 were obtained from Abcam (Cambridge, MA, USA). 4′,6-diamidino-2-phenylindole (DAPI) and d -luciferin bioluminescent substrate were purchased from Sigma-Aldrich (St Louis, MO, USA). Capivasertib and LY294002 were purchased from Selleckchem (Houston, TX, USA).
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