A library of 78 natural compounds was obtained from Target Molecule Corp. Digitoxin (≥98% pure) was purchased from Baoji Herbest Bio-Tech Co., Ltd. MTT was supplied by Sigma-Aldrich (Merck KGaA). An Annexin-V-FITC/propidium iodide (PI) staining assay kit was obtained from Beyotime Institute of Biotechnology. The bicinchoninic protein assay kit (BCA) was purchased from Thermo Fisher Scientific Inc., while PI and 4′,6-dimidyl-2-phenylindole (DAPI) were purchased from Roche Diagnostics (Shanghai) Co. Ltd. Primary antibodies against cyclin-dependent kinase 1 (CDK1, #9116), cyclin B1 (#4138), phosphorylated (p)-CDK1 (Thr14) (#2543), p-histone H2AX (γH2AX, #9718), ATR (#2790), p-ATR (Ser428) (#2853), CHK2 (#6334), p-Chk2 (Thr68) (#2197), CDC25C (#4688), p-CDC25C (Thr48) (#12028), Bax (#5023), Bcl-2 (#15071), cytochrome c (#11940), caspase-9 (#9508) and-3 (#9662), cleaved-caspase-3 (#9579) and −9 (#20750), cleaved poly (ADP-ribose) polymerase (PARP) (#5625), β-actin (#4970) and the horse-radish peroxidase (HRP)-conjugated secondary antibodies (Anti-mouse IgG, #7076; Anti-rabbit IgG, #7074), Alexa Fluor 647-conjugated Anti-rabbit IgG (H+L) (#4414) were obtained from Cell Signaling Technology Inc., (dilution of primary antibodies, 1:1,000; dilution of secondary antibodies, 1:2,000).
P atr
Manufactured by Cell Signaling Technology
Sourced in United States
The P-ATR is a laboratory equipment product that functions as a phospho-specific antibody for the detection of phosphorylated ATR (Ataxia Telangiectasia and Rad3-related) protein. This antibody is designed to specifically recognize and bind to the phosphorylated form of the ATR protein, which plays a crucial role in the cellular response to DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
P atm, by Cell Signaling Technology (20 mentions)
P chk1, by Cell Signaling Technology (17 mentions)
P chk2, by Cell Signaling Technology (15 mentions)
γh2ax, by Cell Signaling Technology (9 mentions)
β actin, by Cell Signaling Technology (6 mentions)
Cleaved parp, by Cell Signaling Technology (6 mentions)
Gapdh, by Santa Cruz Biotechnology (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (5 mentions)
P h2ax, by Cell Signaling Technology (4 mentions)
β actin, by Merck Group (4 mentions)
Caspase 3, by Cell Signaling Technology (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
P brca1, by Cell Signaling Technology (3 mentions)
Bcl 2, by Cell Signaling Technology (3 mentions)
Caspase 9, by Cell Signaling Technology (3 mentions)
Ripa lysis buffer, by Avantor (2 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (2 mentions)
Novex 3 8 tris acetate 15 well mini gels, by Thermo Fisher Scientific (2 mentions)
Immobilon p membrane, by Thermo Fisher Scientific (2 mentions)
34 protocols using p atr
1
Digitoxin Induces Apoptosis Signaling
2
Western Blotting Analysis of DNA Damage Signaling
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After being washed with ice-cold PBS, cells were lysed with RIPA Lysis Buffer (VWRVN653-100ML, VWR Life Science), supplemented with Phosphatase Inhibitor Cocktail 2 (P5726-1ML, Sigma) and Protease Inhibitor Cocktail (B14001, Bimake). The Pierce BCA Protein Assay Kit (89167–794, Thermo Scientific) was used to determine protein concentration. Equal protein lysates were run on Novex 3–8% Tris-acetate 15 Well Mini Gels (EA03785BOX, Invitrogen) and transferred to Immobilon-P membranes (IPVH00010, Fisher Scientific). Membranes were then probed with the following primary antibodies: GAPDH (sc-47724, Santa Cruz Biotechnologies, 1:1000) and phospho DNA-PKcs S2056 (ab18192, Abcam). P300 (sc-48343, Santa Cruz Biotechnologies), pATM (13050S, Cell signaling), ATM (92356S, Cell Signaling), pATR (58014S, Cell signaling), and ATR (2790S, Cell signaling). After exposure to the matching HRP-conjugated secondary antibody, cells were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (34095, Thermo Scientific).
3
Synthesis and Characterization of Anticancer Agents
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MPT0G211, tubastatin A (TBA), and SAHA were synthesized by Dr. Jing-Ping Liou’s Lab. (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan), and the purity are more than 98%. Doxorubicin (DOXO), cyclophosphamide (CTX), and vincristine (VCR) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-Tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA). PARP and CDC2 were from Santa Cruz Biotechnology (Dallas, TX, USA).
4
Evaluating Apoptotic Signaling in Chemoresistant Lung Cancer Cells
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Based on Lee et al. [25 (link)], Western blotting was conducted in A549/CR and H460/CR cells exposed to PGG (0, 6.25, 12.5 and 25 μM) for 48 h. In brief, whole cell lysates were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail, and isolated proteins in the supernatants were quantified and electrotransferred onto a Hybond enhanced chemiluminescence (ECL) transfer membrane. The membranes were probed with primary antibodies for PTEN, p-AKT, PARP, cellular inhibitor of apoptosis 1 (c-IAP1), c-IAP2, Survivin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), Caspase-8, -9, -7, Cleaved caspase-3, BAX, Bcl-2, Bcl-xL, p-ATR, p-Chk2, p-BRCA-1, p-H2AX (Cell signaling Technology, Danvers, MA, USA), p53 (Oncogene Research Products, San Diego, CA, USA), XIAP (Becton Dickinson and Company BD Biosciences, San Jose, CA, USA), β-actin (Sigma Aldrich, St Louis, MO) and horseradish peroxidase-conjugated secondary antibody. Additionally, nuclear extract was isolated for DNA damage proteins using an NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Thermo Scientific, Rochester, NY, USA).
5
Ebselen Inhibits H1N1 Influenza Virus-Induced Apoptosis
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Madin-Darby canine kidney cells (MDCK) were obtained from American Type Culture Collection (ATCC CCL-34TM, Rockville, USA). H1N1 influenza virus was isolated from clinical samples of Guangzhou Women and Children’s Medical Center, Guangzhou Medical University (Guangzhou, China). Fetal bovine serum (FBS), pancreatic enzymes, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco (California, USA). P-ATM, P-ATR, PARP, Cleaved PARP, Caspase-3, Cleaved Caspase-3, and β-actin antibodies were purchased from Cell Signaling Technology (Boston, USA). Cell counting kit-8 (CCK-8 kit), reverse transcription-polymerase chain reaction kit (RT-PCR kit), JC-1 Mitochondrial Membrane Potential Assay Kit, Cell Cycle Assay Kit, Annexin-V-Propidium iodide (PI) Co-staining Kit, and Enhanced Chemiluminescence (ECL) Assay Kit were obtained from Beyotime Biotechnology (Shanghai, China). Tunel, 4′, 6-diamidino-2-phenylindole (DAPI), and 2′, 7′-dichlorofluorescein diacetate (DCF-DA) were purchased from Sigma-Aldrich (St. Louis, USA). The detection of the cells and the cytokine were derived from the BD FACSCanto II flow cytometer (Franklin Lakes, USA). Ebselen was provided by Tianfeng Chen, College of Chemistry and Materials Science, Jinan University (Guangzhou, China).
6
Western Blot Analysis of DNA Damage Response
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Protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, UK). Blots were blocked for 1 h in 5% milk/0.1% Tween 20 in phosphate buffered saline (PBS-T) and then incubated with primary antibodies (1: 1000) at 4°C overnight. Blots were then washed three times for 15 min in PBS-T, followed by incubation with secondary antibody (according to different primary antibodies, HRP-conjugated goat anti-mouse, anti-rabbit, and rabbit anti-goat IgG were used (1: 5000, Santa Cruz, Dallas, TX)) in 5% milk/PBS-T for 1 h, and then washed three times for 15 min in PBS-T. The membranes were briefly incubated with ECL detection reagent (Amersham Biosciences, Castle Hill, Australia) to visualize the proteins and were then exposed on X-ray film. Primary antibodies used were as follows: ATM (Cat# 600-401-398), and p-ATM (Cat# 600-401-400) were purchased from Rockland Immunochemical (Gilbertsville, PA, USA); cleavage-caspase-3 (Cat# 9661), cleavage-PARP (Cat# 5625P), γ-H2Ax (Cat# 9718), Bax (Cat# 2772s), and Bcl-2 (Cat# 2870), ATR (Cat# 2790), p-ATR (Cat# 2853S) were from Cell Signaling Technology (Beverly, MA); PARP (Cat# 7150), pro-caspase-3 (Cat# 7272), β-Actin (Cat# 1615), Chk1 (Cat# 377231), p-Chk1 (Cat# 2341), Chk2 (Cat# 8813), p-Chk2 (Cat# 2661) were from Santa Cruz (Dallas, Texas, US).
7
Protein Expression Analysis of HH32 and HH33
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The protein expression analysis was done after treatment with 0.5 or 1μM of HH32 and HH33 compounds for 24 h by Western blot.26 Cell lysates were prepared using lysis buffer containing 20% SDS, glycerol, 1M Tris (pH 6.8) and supplemented with protease and phosphatase inhibitors (Sigma Aldrich, Missouri, USA). The proteins were separated by SDS-PAGE and transblotted into nitrocellulose membrane (Biorad, Hercules, CA, USA). Membrane were incubated overnight at 4°C with the following primary antibodies: γH2AX, H2Ax, p21, CDC2, CDC25c, cyclin B, c-Myc, Bid, ATM, p-ATM, ATR, p-ATR, Chk1, p-Chk1, Chk2, p-Chk2, caspase 9, caspase 8, pRb (Ser807/811) (Cell Signaling Technology, Massachusetts, USA), p53, cyclin A, cyclin E, cyclin D1, CDK2, Rb, pRb (Ser249) (Santa Cruz Biotech, Texas, USA) and β-actin (Sigma Aldrich, Missouri, USA). Membranes were then incubated with respective mouse/rabbit secondary antibody (Cell Signaling Technology, Massachusetts, USA) for 1 h at room temperature and detected by enhanced chemiluminescence (ECL) (Biorad, Hercules, CA, USA) method using Chemi Doc imaging system (Biorad, Hercules, CA, USA). The protein bands were quantified using Image lab software (Biorad, Hercules, CA, USA).
8
Detecting DNA Damage Response Proteins
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The proteins from the cultured cells were harvested by using a phenylmethylsulfonyl fluoride (PMSF) lysate buffer. Samples containing 50 μg of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to polyvinylidene fluoride membranes. After the membranes were blocked with 5% non-fat milk for 1 h, they were incubated with the primary antibody at 4 °C overnight: NEIL3 (ab230908; Antibody Cambridge (Abcam)), p-ATR (2853S; Cell Signaling Technology (CST)), p-ATM (5883S; CST), γ-H2AX (9718S; CST), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118S; CST). The membranes were then incubated with the horseradish peroxidase (HRP)-labeled secondary antibody (Proteintech, Wuhan, Hubei, China) at 37 °C for 1 h. The protein band signal was detected using the BeyoECLPlus chemiluminescence reagent (Beyotime, Shanghai, China).
9
Antibodies for DNA Damage Response
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Antibodies (poly [ADP‐ribose] polymerase [PARP], cleaved PARP, cleaved caspase 3, pH2AX, Chk1, p‐Chk1, Chk2, p‐Chk2, ATM, p‐ATM, ATR, p‐ATR, MRE11, NBS1, RAD50, and glyceraldehyde 3‐phosphate dehydrogenase [GAPDH]) used for Western blot analysis were purchased from Cell Signaling Technology (Danvers, MA, USA). KLF5 antibody was purchased from Abcam (ab137676; Cambridge, MA, USA). Cisplatin was purchased from Sigma‐Aldrich (St. Louis, MO, USA) and dissolved in 0.9% NaCl solution.
10
Caspase Activation Pathway Monitoring
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TG emulsion LipofundinⓇ MCT/LCT 20% (B. Braun Melsungen AG, Melsungen, Germany) was used to deliver TG into cells as described previously (36 (link)). Caspase-1 substrate Ac-YVAD-p-nitroanilide (Ac-YVAD-pNA) was purchased from Biomal (Plymouth Meeting, PA, USA). Caspase-2 substrate Ac-VAVAD-pNA and RNase A were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against cleaved caspase-8, cleaved caspase-3, cleaved caspase-7, PARP, p-ATM, p-ATR, p-Chk1, p-Chk2, and p-H2AX were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against β-actin and the caspase-2-specific inhibitor z-VDVAD-FMK were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The caspase-8-specific inhibitor z-IETD-FMK was obtained from R&D Systems (Minneapolis, MN, USA). The ATM/ATR kinase inhibitor CGK733 was purchased from Calbiochem (Darmstadt, Germany).
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