After being irradiated, the cells were incubated for 24 h and then lysed with protease inhibitor. Aliquots of cell lysates were loaded at adequate amounts onto gels and then subjected to electrophoresis. Samples was then transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Burlington, MA, USA) and further incubated with primary antibodies to phosphorylated ATM (pATM, GeneTex, Irvine, CA, USA), poly (ADP-ribose) polymerase-1 (PARP1, Cell Signaling Technology, Danvers, MA, USA), γ-H2AX (GeneTex), and β-tubulin (Merck Millipore, Burlington, MA, USA). Bound antibodies were detected using appropriate peroxidase-coupled secondary antibodies followed by enhanced chemiluminescence (ECL, Boehringer Mannheim, Mannheim, Germany). Images were obtained after washing out the secondary antibody. The fluorescence images were visually scanned for luminescence and quantitatively analyzed using ImageJ software.
Poly adp ribose polymerase 1 parp1
Manufactured by Cell Signaling Technology
Sourced in United States
Poly (ADP-ribose) polymerase-1 (PARP1) is an enzyme that catalyzes the addition of poly(ADP-ribose) chains to target proteins. It plays a key role in various cellular processes, including DNA repair, transcriptional regulation, and cell death.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3, by Cell Signaling Technology (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
Cleaved parp 1, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
γ h2ax, by GeneTex (1 mentions)
Enhanced chemi luminescence (ecl), by Roche (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
β tubulin, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Xestospongin c, by Bio-Techne (1 mentions)
Ym 58483, by Merck Group (1 mentions)
Phospho perk, by Santa Cruz Biotechnology (1 mentions)
Antibodies specific for, by Santa Cruz Biotechnology (1 mentions)
Caffeine, by Merck Group (1 mentions)
Methylglyoxal, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Control antibody, by Santa Cruz Biotechnology (1 mentions)
12 protocols using poly adp ribose polymerase 1 parp1
1
Quantifying DNA Damage Response Proteins
2
Investigating Apoptotic Signaling in HCT116 Cells
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For Western blot analysis, single-cell suspensions of HCT116 cells were plated (0.5 x 106 cells per 60 mm dish) in triplicate. After 24 h, once the cells were attached to the plates, they were treated with small molecule inhibitor(s) alone or in combination with TMZ for 48 h. Changes in protein levels subsequent to the treatment of SMI’s were determined by Western blot analysis using whole-cell extracts. The antibodies used to detect the levels of p53, p21, Bcl2, Bax, Poly [ADP-ribose] polymerase 1 (PARP-1), cleaved PARP1, cleaved caspase 3, caspase 3, apoptosis inducing factor (AIF) and GAPDH were obtained from Cell Signaling Technologies (Danvers, MA).
3
Methylglyoxal-Induced Cell Stress Responses
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Methylglyoxal, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), N‐acetylcysteine (NAC), 4‐phenyl butyric acid (4‐PBA), MRS1845, YM‐58483, caffeine, dichlorodihydrofluorescein diacetate (H2DCFDA), dihydroethidium (DHE) and propidium iodide (PI) were obtained from Sigma‐Aldrich Co (St Louis, MO, USA). Xestospongin C was purchased from Tocris Bioscience (Bristol, UK). Z‐Val‐Ala‐Asp‐fluoromethylketone (z‐VAD‐FMK), salubrinal and 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetrakis/acetoxymethyl ester (BAPTA/AM) were purchased from Calbiochem (Darmstadt, Germany). The antibodies specific for caspase‐3, caspase‐9, eIF2α, phospho‐eIF2α and poly(ADP‐ribose) polymerase 1 (PARP1) were purchased from Cell Signalling Technology (Beverly, MA, USA). The antibodies specific for glucose‐regulated protein 78 (GRP78), CHOP, PERK, phospho‐PERK and β‐actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies specific for ATF4, ATF6, IRE1β and XBP1 were purchased from Abcam (Cambridge, UK).
4
Cytotoxic Effects of Ptac2S and CDDP
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Ptac2S was prepared according to previously reported procedures [4 (link), 5 (link)]. CDDP was purchased from Sigma-Aldrich, Chemicals (Milan, Italy). RPMI 1640 medium, antibiotics, glutamine and foetal bovine serum were purchased from Celbio (Milan, Italy). Caspase -9, -7 and -3, Bax, Bcl-2, poly(ADP-ribose) polymerase-1 (PARP-1), were obtained from Cell Signalling Technology (Celbio, Milan, Italy). PKC isoforms antibodies, phospho-specific p-38MAPK and total (phosphorylated and unphosphorylated) p-38MAPK antibodies, anti-porin (i.e. anti-voltage-dependent anion selective channel 1, VDAC1), goat anti-rabbit conjugated with peroxidase, as well as control antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All others reagents were from Sigma-Aldrich (Milan, Italy).
5
Mechanistic Study of SLC1A1 Inhibition
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SN38 was from Sigma-Aldrich, oxaliplatin from Sanofi-Aventis, and DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA) from Tocris. UCPH101 was synthesized as described [29 (link)]. Primary antibodies were from Santa Cruz Biotechnology (SLC1A1, p21WAF1/Cip1 (p21) and CTR1), BD Transduction (p150), Cell Signaling Technology (poly-(ADP-ribose) polymerase-1 (PARP-1), phospho-Ser807/811-retinoblastoma protein, and p53). Rhodamine-phalloidin was from Invitrogen, and AlexaFluor488-conjugated secondary antibody from Life Technologies. 4',6-diamidino-2-phenylindole (DAPI) was from Invitrogen. Alkaline phosphatase-coupled secondary antibodies were from Sigma-Aldrich. [3H]-D-Aspartic acid ([3H]-D-Asp) was from PerkinElmer.
6
Investigating Apoptosis and Autophagy Regulation in Cellular Responses
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Fetal bovine serum (FBS), RPMI-1640, and penicillin-streptomycin were purchased from HyClone (Victoria, Australia). 3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) and JC-1 fluorescent dye (Sigma-Aldrich, St Louis, MO), LY294002 and SP600125 (selleck, Texas, Houston), Protein A/G plus Agarose immunoprecipitation reagent (Santa Cruz, CA), DAPI (C0060, Solarbio, Beijing, China), lysis buffer (Beyotime, Shanghai, China), Annexin- V/FITC Apoptosis Detection Kit (BestBio, Shanghai, China). Antibodies specific for β-actin (sc-1615), Bax (sc-493), Bcl-2 (sc-783), Nrf2 (sc-13,032), c-Jun (sc-74,543) and p-c-Jun (sc-53,182) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). p-Nrf2 (ab76026), p62 (ab56416), Anti-active caspase 3 (ab2302) and anti-active caspase 9 (ab2324) were purchased from Abcam (Cambridge, MA). SAPK/JNK siRNA (#6232), p62 siRNA (#6394), Poly (ADP-ribose) polymerase-1 (PARP-1) (#9532), Cleaved PARP (#5625), NQO1 (#3187), Keap1 (#4617), Bcl-xL (#2764), XIAP (#14334), JNK (#9252), p-JNK (#9255), LC3B (#3868S) and Autophagy antibody sampler kit (#4445) including Beclin-1, ATG16, ATG3, ATG7 and ATG5 were purchased from Cell Signaling Technology (Danvers, MA). The enhanced chemiluminescence (ECL) kit was purchased from ThermoFisher (Waltham, MA).
7
Histone Deacetylase and Apoptosis Pathways
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CKD-581 was provided from CKD Pharmaceutical Corp. (Seoul, Korea) and suberoylanilide hydroxamic acid (SAHA, vorinostat) with a purity of >98% was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rituximab was purchased from Roche (Basel, Switzerland). Compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. The CellTiter-Glo luminescent cell viability assay kit was supplied from Promega (Madison, WI, USA). Antibodies recognizing histone H3, acetylated histone H3, tubulin, acetylated tubulin, myeloid cell leukemia-1 (MCL-1), and poly (ADP-ribose) polymerase 1 (PARP1) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies for BCL-2, BCL-6, and BCL-XL were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibody for phosphorylated H2AX (γH2AX) was purchased from Abcam (Cambridge, MA, USA). Antibodies for MYC, CD20, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were supplied from Thermo Fisher Scientific (Waltham, MA, USA).
8
Quercetin-Mediated Apoptosis Induction
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Quercetin, dexamethasone, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), propidium iodide (PI), and ribonuclease A were all purchased from Sigma(St. Louis, MO, USA) Z-VAD-FMK was purchased from R&D systems (USA). Annexin V Apoptosis Detection Kit FITC/PI was obtained from eBioscience (CA, USA). Primary antibodies against caspase-3, caspase-8, caspase-9, poly(ADP-ribose)polymerase 1 (PARP-1), and c-myc were procured from Cell Signaling Technology (MA, USA). Primary antibody against p21 was obtained from Abcam (Cambridge, UK). Primary antibody against β-actin was obtained from Sigma-Aldrich. Horseradish peroxidase (HRP)–conjugated antirabbit and antimouse antibodies were procured from Jackson Immuno Research Laboratories (PA, USA).
9
Protein Expression Analysis by Western Blot
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Western blot analysis was performed as previously described [16 ]. Primary antibodies against Bax (rabbit, diluted 1:1000), poly(ADP-ribose) polymerase 1 (PARP1) (rabbit, diluted 1:1000), caspase-3 (rabbit, diluted 1:1000), β-catenin (rabbit, diluted 1:1000), phosphorylated (P)-JNK (rabbit, diluted 1:1000), and total-JNK (T-JNK) (rabbit, diluted 1:1000) were purchased from Cell Signaling Technology. Antibodies against sFRP1 (rabbit, diluted 1:500) were obtained from Cloud-Clone Corp. (CCC, USA). Primary antibodies against α-tubulin (diluted 1:5000, Sigma, USA) and glyceraldehyde 3-phosphate dehydrogenase (diluted 1:1000, Sigma, USA) served as loading controls for whole-cell lysates. The band intensity was analyzed by Quantity One software (Bio-Rad, USA).
10
Comprehensive Antibody Profiling for Apoptosis and ER Stress
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The antibodies against procaspase-3, procaspae-9, procaspase-8, Bax, Bad, Bcl-xL and Bcl-2, apoptosis-inducing factor (AIF), poly(ADP-ribose) polymerase-1 (PARP-1) and C/EBP homologous protein (CHOP) were acquired from Cell Signaling Technology (Danvers, MA, USA). The antibodies against eukaryotic translation initiation factor 2 alpha (eIF2α), phospho-eIF2α, activating transcription factor 4 (ATF4), phospho-protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), and inositol-requiring enzyme 1 alpha (IRE1α) were from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The antibodies against cleaved caspase-3, cleaved caspase-9, and β-actin were from EMD Millipore (Bellerica, MA, USA). The antibodies against activating transcription factor 6 (ATF6) alpha were from Abcam (Cambridge, MA, USA). The antibodies against cytochrome c (cyt c) were from Proteintech Group (Chicago, IL, USA). The secondary horseradish peroxidase-conjugated IgG was from GeneTex (Irvine, CA, USA). Protease inhibitor cocktail was obtained from Sigma-Aldrich Co. (St Louis, MO, USA). Polyvinylidene difluoride membranes and Chemiluminescent horseradish peroxidase substrate were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
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