Cleaved caspase 3 asp175 5a1e rabbit mab

Manufactured by Cell Signaling Technology

Sourced in United States

Cleaved caspase-3 (Asp175) (5A1E) rabbit mAb is a laboratory reagent that specifically recognizes the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. Caspase-3 is a key effector of apoptosis and is activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways.

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5 protocols using cleaved caspase 3 asp175 5a1e rabbit mab

Elesclomol Induces Apoptosis in Rabbit Cells

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New Zealand white rabbits, weighing 2.2–2.7 kg each, were purchased from the Hushan Experimental Animal Center (License no.: SCXK 2015-0004), Wuxi, China. The animal experiments were approved by the Experimental Animal Committee of Jiangnan University. Elesclomol (STA-4783) was obtained from Selleck Chemicals (Houston, TX, USA), dissolved in dimethyl sulfoxide (DMSO; Beyotime, Shanghai, China), and stored at −20 °C. Anti-β-actin antibody (ab16039) was purchased from Abcam (Cambridge, MA, USA). Cleaved caspase-3 (Asp175; 5A1E) rabbit mAb (#9664) was purchased from Cell Signaling Technology (Danvers, MA, USA). Caspase-3 rabbit antibody (19677-1-AP) was purchased from Proteintech (Chicago, IL, USA). 2,7-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Sigma-Aldrich (D6883; St. Louis, MO, USA), and the Annexin V apoptosis-detection kit was obtained from Thermo Fisher Scientific (BMS500BT-100; Waltham, MA, USA). Recombinant human transforming growth factor (TGF)-β1 was purchased from PeproTech (#0212209; Rocky Hill, NJ, USA).

Feng Y., Wu J.J., Sun Z.L., Liu S.Y., Zou M.L., Yuan Z.D., Yu S., Lv G.Z, & Yuan F.L. (2020). Targeted apoptosis of myofibroblasts by elesclomol inhibits hypertrophic scar formation. EBioMedicine, 54, 102715.

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Chromatographic Separation and MS Analysis

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Flash column chromatographic separations were accomplished on Biotage Isolera Four system (Biotage, Uppsala, Sweden). NMR was recorded on DRX-400 and DRX-500 NMR spectrometers (Bruker, Rheinstetten, Germany). ESI-, APCI- and HR ESI-MS were recorded on Agilent 1290 LC-MS or Agilent G6530 TOF MS spectrometer (Agilent Technologies Inc., California, USA). Western blot results were visualized on Mini-HD9-Auto Biomolecular imager (Leader Oriental Technology. LTD, Beijing, China). Dichlone and flavonoids were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and Ark Pharm, Inc. (Arlington Heights, IL, USA). Cell lines were gotten from the American Type Culture Collection (Manassas, VA). Caspase-3 (3G2) mouse mAb, caspase-3 antibody, cleaved caspase-3 (Asp175) (5A1E) rabbit mAb, PARP (46D11) rabbit mAb and horseradish peroxidase (HRP) were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA).

Luo P., Wei W., Haider S., Khan S.I., Wang M., Pan W, & Chittiboyina A.G. (2020). Synthesis of benzonaphthofuroquinones and benzoylnaphthindolizinediones by reactions of flavonoids with dichlone under basylous, oxygenous and aqueous conditions: their cytotoxic and apoptotic activities. RSC Advances, 10(48), 28644-28652.

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Immunoblotting Analysis of Signaling Proteins

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Antibodies used were anti-phospho-AKT(Ser 473), anti-phospho-AKT(Thr308), and anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit mAb (Cell Signaling Technology, Danvers, MA), anti-Akt (pan) Rabbit mAb, p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb(Cell Signaling Technology, Danvers, MA), anti-PARP rabbit mAb and Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (Cell Signaling Technology, Danvers, MA), anti-alpha tubulin monoclonal horseradish peroxidase conjugate (Cell Signaling Technology, Danvers, MA), Secondary antibodies (horseradish peroxidase-linked anti-rabbit IgG and anti-mouse IgG antibodies) were obtained from Amersham Biosciences (Uppsala, Sweden).

Majlessipour F., Zhu G., Baca N., Kumbaji M., Hwa V, & Danielpour M. (2024). Skeletal overgrowth in a pre-pubescent child treated with pan-FGFR inhibitor. Heliyon, 10(11).

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Western Blot Analysis of Protein Biomarkers

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The cell lysates were homogenized in mammalian protein extraction buffer (Sigma). The resulting lysates were centrifuged at 12,000 rpm, and 4°C for 15 min, and the protein contents were measured using a protein-assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA). Subsequently, 10–15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis was performed, followed by transfer onto nitrocellulose blotting membranes (Amersham, GE Healthcare Life Science, Germany). The antibodies used for immunoblotting are as follows: Bax rabbit mAb (1:1,000; Cell Signaling Technology), Bcl2 (D17C4) rabbit mAb (1:1,000; Cell Signaling Technology), inducible nitric oxide synthase (iNOS; NOS2) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), AMPKα (D63G4) rabbit mAb (1:1,000; Cell Signaling Technology), Phospho-AMPKα (Thr172) (D4D6D) rabbit mAb (1:1,000; Cell Signaling Technology), caspase-3 rabbit mAb (1:1,000; Cell Signaling Technology), cleaved caspase-3 (Asp175) (5A1E) rabbit mAb (1:1,000; Cell Signaling Technology), PARP (46D11) rabbit mAb (1:1,000; Cell Signaling Technology), and β-actin (13E5) rabbit mAb (1:2,500; Cell Signaling Technology). The protein bands were visualized using an enhanced chemiluminescence method with an ELC kit (Millipore, Billerica, MA, USA) and were quantified using Quantity 1 v.4.6.7 (Bio-Rad).

Kim K., Kwak M.K., Bae G.D., Park E.Y., Baek D.J., Kim C.Y., Jang S.E., Jun H.S, & Oh Y.S. (2021). Allomyrina dichotoma larva extract attenuates free fatty acid-induced lipotoxicity in pancreatic beta cells. Nutrition Research and Practice, 15(3), 294-308.

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Immunocytochemistry Staining of MuSCs

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Immunocytochemistry staining was performed on MuSCs after fixation and permeabilization.
MuSCs were fixed with 4% PFA for 10 minutes at room temperature then permeabilized using 0.3% Triton-X PBS for 15 minutes. Cells were then blocked with 10% donkey serum in 0.3% Triton-X PBS for 15 minutes. Cleaved caspase-3 was probed in MuSCs by incubating the cells overnight in 1:100 Cleaved Caspase-3 (Asp175) (5A1E) rabbit mAb (Cell Signaling Technology). MuSCs were stained for 3 hours with 1:500 donkey anti-rabbit Alexa 594 secondary antibody (Invitrogen) to detect cleaved caspase-3. pRB was stained for in MuSCs following EdU staining. MuSCs were first permeabilized then blocked in donkey serum for 20 minutes. After blocking, the cells were stained using 1:100 phospho-Rb (Ser807/811) (D20B12) XP rabbit mAb (Cell Signaling Technology). Secondary staining was performed using 1:500 donkey anti-rabbit Alexa 488 antibody (Invitrogen). All primary and secondary antibodies were diluted in 10% donkey serum.

Ahsan S., Raval M.H., Ederer M., Tiwari R.L., Chareunsouk A., & Rodgers J.T. (2020). Metabolism of glucose and glutamine is critical for skeletal muscle stem cell activation.

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