Cleaved caspase 3 asp175

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom, Germany

Cleaved caspase-3 (Asp175) is a laboratory reagent used to detect the presence and activation of the apoptotic protein caspase-3. It is a specific antibody that recognizes the cleaved form of caspase-3, which is a key executioner of apoptosis. This product can be used in various analytical techniques, such as Western blotting and immunohistochemistry, to study the role of caspase-3 in cellular processes.

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Cleaved caspase-3 (3596 citations) Caspase-3 (2138 citations) Asp175 (134 citations) Rabbit anti-Cleaved Caspase-3 (Asp175) (35 citations) Anti-cleaved caspase-3 (Asp175) antibody (13 citations) PathScan Cleaved Caspase-3 (Asp175) sandwich ELISA kit (7 citations) Caspases (2 citations) SignalStain cleaved Caspase 3 (Asp175) IHC Detection kit (2 citations) Rabbit-antihuman cleaved caspase-3 (Asp175) (2 citations) Anti-cleaved caspase 3 (Asp175) (9664) (2 citations)

151 protocols using cleaved caspase 3 asp175

Western Blot Analysis of Oxidative Stress Markers

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After rinsing in cold PBS three times, the cells were homogenized in RIPA buffer. The supernatant was then centrifuged at 120,000×g for 15 min at 4 °C. The samples (10–20 mg) were run on SDS-PAGE gels, transferred to PVDF filter membranes, and used for western blotting with monoclonal antibodies against CaMKIIδ (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p47^phox, Nox2/gp91^phox (Abcam, Cambridge, MA, USA), cleaved caspase-3 (Asp175) (Cell Signaling Technology), and ApoJ (EterLife, Birmingham, UK). The PVDF membranes were then incubated with HRP-conjugated anti-rabbit immunoglobulin G antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. The blot was developed with an ECL-Plus chemiluminescence reagent kit and visualized with the UVP Bio-Imaging System. The blot densities were analyzed using Image J.

Ma Y., Gong Z., Nan K., Qi S., Chen Y., Ding C., Wang D, & Ru L. (2019). Apolipoprotein-J blocks increased cell injury elicited by ox-LDL via inhibiting ROS-CaMKII pathway. Lipids in Health and Disease, 18, 117.

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Quantifying Apoptosis in Cerebellar Tissue

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Animals were sacrificed via cervical dislocation. The cerebella were immediately immersed in 4% PFA for 24 hr and embedded in paraffin. Five micrometer sagittal sections were cut using a microtome (RM2255, Leica). Following deparaffinization, epitope retrieval was performed by boiling the sections in 0.1M citrate buffer (pH 6). The sections were then blocked and permeabilized in 1% albumin, 0.1% Triton X‐100, and 0.05% Tween‐20. Sections were incubated with primary antibody against cleaved caspase 3 Asp175 (Cell Signaling, 9661; 1:500) O/N at 4°C, and with secondary biotinylated anti‐rabbit antibodies (Vector, BA‐1000). The staining was visualized using the ABC Kit Vectastain Elite and DAB substrate (Vector Laboratories). Three animals at each time‐point were used for each genotype. Positively stained cells were manually counted from 3 to 4 cerebellar sections/animal.

Murru S., Hess S., Barth E., Almajan E.R., Schatton D., Hermans S., Brodesser S., Langer T., Kloppenburg P, & Rugarli E.I. (2019). Astrocyte‐specific deletion of the mitochondrial m‐AAA protease reveals glial contribution to neurodegeneration. Glia, 67(8), 1526-1541.

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Comprehensive Lung Tissue Analysis Protocol

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Lung tissue samples were fixed in 2% formaldehyde overnight and following dehydration in 100% ethanol embedded in paraffin. 5 μm thick sections were stained by hematoxylin and eosin (H&E). Slides were scanned and tumor area, tumor number and tumor grade were determined in a blinded manner, using TissueFaxs and HistoQuest (TissueGnostics) software, respectively. Tumor grades were classified by board certified pathologists HP, LB, KD and JM.(30 (link)) For immunohistochemistry (IHC) and immunofluorescence (IF), 5 μm sections were subjected to stainings using standard protocols and antibodies against: EGFR (1:300, BD Biosciences #610016), Ki-67 (1:400, Cell Signaling#12202), phospho-Erk1/2 (Thr202/Tyr204; 1:400, Cell Signaling#4370), Cleaved Caspase 3 (Asp175; 1:200, Cell Signaling#9661) and phospho-Akt (Ser473; 1:100, Cell Signaling#3787). For analysis, at least 5 different high power field sections per group were compared, using HistoQuest for IHC and TissueQuest (TissueGnostics) for IF, respectively, as previously described(20 ).

Moll H.P., Pranz K., Musteanu M., Grabner B., Hruschka N., Mohrherr J., Aigner P., Stiedl P., Brcic L., Laszlo V., Schramek D., Moriggl R., Eferl R., Moldvay J., Dezso K., Lopez-Casas P.P., Stoiber D., Hidalgo M., Penninger J., Sibilia M., Győrffy B., Barbacid M., Dome B., Popper H, & Casanova E. (2018). Afatinib restrains K-RAS driven lung tumorigenesis. Science translational medicine, 10(446), eaao2301.

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Immunohistochemical Analysis of Lung Tissue

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Lungs were perfused with 10% formalin, stored in fixative overnight, and embedded in paraffin. For further staining (hematoxylin/eosin and immunohistochemisty), sections of 5 μm were cut. Primary antibodies from Cell Signaling Technologies (Danvers, MA, USA) were used at the listed dilutions: phospho-Histone H2A.X (Ser139) (#20E3) (1:400), Cleaved Caspase-3 (Asp175) (#5A1E) (1:800). The Ki67 antibody (16A8) was purchased from BioLegend (San Diego, CA, USA) and used at a dilution of 1:50. Positive cells were identified visually in each high-power field (40× magnification). A total of 3–5 fields were observed per tumour.

Herter-Sprie G.S., Korideck H., Christensen C.L., Herter J.M., Rhee K., Berbeco R.I., Bennett D.G., Akbay E.A., Kozono D., Mak R.H., Makrigiorgos G.M., Kimmelman A.C, & Wong K.K. (2014). Image-guided radiotherapy platform using single nodule conditional lung cancer mouse models. Nature communications, 5, 5870.

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Lung Nodule Protein Profiling

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Lung nodules were homogenized, lysed using RIPA (Santa Cruz, TX), and 10% SDS/PAGE was run after protein quantification (Bio‐Rad, Hercules, CA). Primary antibodies (1:1000): PD‐L1, p‐Stat3‐705, Stat‐3, phospho‐p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, cleaved caspase‐3 (Asp175), and caspase‐3 (Cell Signaling, MA) were used. Autoradiography detection and quantification were performed using Image J.

Dhupkar P., Gordon N., Stewart J, & Kleinerman E.S. (2018). Anti‐PD‐1 therapy redirects macrophages from an M2 to an M1 phenotype inducing regression of OS lung metastases. Cancer Medicine, 7(6), 2654-2664.

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Histopathological Characterization of Tissues

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Tissues were fixed in 10% formalin, paraffin embedded and analyzed on hematoxylin and eosin (H&E) stained sections. Immunohistochemistry was performed using Vectastain Elite ABC and DAB Substrate kits (Vector Laboratories). Methanol/hydrogen peroxide pretreatment, microwave 10 mmol/L citrate antigen retrieval, and serum blocking was performed. Antibodies were incubated at 4°C overnight: Calcitonin (1:8000; Bachem), p53 (1:500; (CM5) Leica Microsystems), phosphorylated histone H3 (1:1000; US Biological) and cleaved caspase-3 (Asp 175) (1:150, Cell Signaling). Slides were counterstained with nuclear fast red. Cell proliferation and apoptosis was quantified by determining percentage positive cells with 303-534 cells analyzed per group.

Akeno N., Miller A.L., Ma X, & Wikenheiser-Brokamp K.A. (2014). p53 suppresses carcinoma progression by inhibiting mTOR pathway activation. Oncogene, 34(5), 589-599.

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Amyloid-beta Immunostaining Protocol

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Antibodies were used that recognized Aβ_l–42(rabbit polyclonal; Cell Signaling; catalog no. 2454) at dilutions of 1:300 to
1:1,000, Aβ_l–16 (Covance; clone 6E10, mouse monoclonal)
at 1:300 to 1:1,000, NG2 (mouse monoclonal and rabbit polyclonal; EMD Millipore;
catalog no. AB5320 and AB5384) at 1:100, MBP (rat monoclonal; EMD Millipore;
catalog no. MAB386) at 1:200, OHg2 (IBL Immuno-Biological Laboratories Co;
catalog no. 18953) at 1:100, Ibal (Wako Life Science; catalog no.
019–19741) at 1:100, GFAP (mouse monoclonal; BD Pharmingen; catalog no.
556329) at 1:500, p21^CDKN1A (Santa Cruz Biotechnology; catalog no.
sc397) at 1:100, CNP (mouse monoclonal; Santa Cruz Biotechnology; catalog no.
sc-166063) at 1:100, pl6 (mouse monoclonal; Abeam; catalog no. ab54210) at
1:100, LAMP1 (rat monoclonal clone 1D4B; Developmental Studies Hybridoma Bank,
University of Iowa and Santa Cruz Biotechnology Inc.; sc-19992) at 1:10 to
1:200, interleukin-6 (Cell Signaling Technology; catalog no. 12912) at 1:100,
cleaved caspase-3 (Asp¹⁷⁵) (Cell Signaling; catalog no. 9661) at
1:1,000. The dye FSB was purchased from EMD Millipore and used at a dilution of
1:1,000. References to prior studies using these antibodies are cited in the
text and/or are available at the vendor’s website.

Zhang P., Kishimoto Y., Grammatikakis L., Gottimukkala K., Cutler R.G., Zhang S., Abdelmohsen K., Bohr V.A., Sen J.M., Gorospe M, & Mattson M.P. (2019). Senolytic therapy alleviates Aβ-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer’s disease model. Nature neuroscience, 22(5), 719-728.

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Protein Expression Profiling by Western Blot

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Protein concentration was measured by the Bicinchoninic Acid (BCA) protein assay (Thermo Fisher Scientific, Rockford, IL, USA). Proteins (30 μg) were separated on 4–20% gradient gels using SDS-PAGE and blotted onto a nitrocellulose membrane. Membranes were blocked in 3% milk in phosphate-buffer saline (PBS) for 2 h and incubated overnight with primary antibodies at 4 °C. The following antibodies were used for immunoblotting: rabbit monoclonal to PPM1D/WIP1 (Cell Signaling, #11901, clone D4F7), p53-pS15 (Cell Signaling, #12571), p21 (Abcam, #109199), KAP1-pS824 (Novus Biologicals, #NB100-2350), γH2AX-pS319 (Cell Signaling, #9718), p53 (Leica-CM5P-L), H2AX (Millipore, 07-627), Cleaved Caspase-3-Asp175 (Cell Signaling, #9664, clone 5A1E), KAP1 (GeneTex, Irvine, CA, USA, #102227, clone N1N2) and Importin β (Santa Cruz, sc-137016). After the washing, membranes were incubated with HRP-conjugated secondary antibodies (BioRad) and signal was detected using Enhanced chemiluminiscence (ECL) Western Blotting Substrate (Thermo Fisher Scientific, Rockford, IL, USA).

Martinikova A.S., Burocziova M., Stoyanov M, & Macurek L. (2020). Truncated PPM1D Prevents Apoptosis in the Murine Thymus and Promotes Ionizing Radiation-Induced Lymphoma. Cells, 9(9), 2068.

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ALCAR Induces Apoptosis in Prostate Cells

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ALCAR ability to induce apoptosis in PCa and BPH cells was confirmed by western blotting. Following 24 h of treatment with ALCAR (1 or 10 mM), cells were lysed in RIPA buffer, supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics GmbH). Proteins (30 μg) were separated on the NupageNovex on 4–12% Bis-Tris Gel (Life Technologies) and transferred to a PVDF membrane Amersham Hybond (GE Healthcare Biosciences). Membranes were incubated overnight at 4 °C with Cleaved Caspase-3 (Asp175) (Cell Signalling Technology) and with peroxidase-linked anti-rabbit IgG or anti-mouse IgG secondary antibodies (GE Healthcare Life science) for 1 h at room temperature. Specific protein bands were detected with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific). Protein expressions were normalized to beta-Actin (Abcam). Band intensity (revealed as optical density-OD) were detected by ImageJ software.

Baci D., Bruno A., Cascini C., Gallazzi M., Mortara L., Sessa F., Pelosi G., Albini A, & Noonan D.M. (2019). Acetyl-L-Carnitine downregulates invasion (CXCR4/CXCL12, MMP-9) and angiogenesis (VEGF, CXCL8) pathways in prostate cancer cells: rationale for prevention and interception strategies. Journal of Experimental & Clinical Cancer Research : CR, 38, 464.

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Immunohistochemical Analysis of Lung Cancer

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Tissue isolation, fixation and staining procedures were performed as previously described in Shackelford et al., 2013. Briefly, the following antibodies were used: phospho-4E-BP1 (Thr37/46) (Cell Signaling Technology, #2855 1:1600), cleaved caspase-3 (Asp175) (Cell Signaling Technology, #9661 1:200), anti-p63 (4A4) (abcam, #ab111449 1:100), anti-TTF-1 (8G7G3/1) (Dako, 1:200), anti-Ki67 (SP6) (Thermo Scientific, #RM-9106-S0 1:200), phospho-S6 (Cell Signaling Technology, #4585 1:400), hexokinase II (Cell Signaling Technology, #2867 1:800), phospho-AMPKα (Thr172) (40H9) (Cell Signaling Technology, #2535 1:100), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP (Cell Signaling Technology, #4370 1:400), phospho-GSKα/β (Ser21/9) (Cell Signaling Technology, #9331 1:50). Immunostained slides were digitally scanned onto a ScanScope AT (Aperio Technologies, Inc., Vista, CA). A pathologist blindly evaluated the H&E stained sections for distribution of histological subtypes, verified by p63 and TTF-1 immunostaining, and calculated the percentage of positively stained cells for Ki67, cleaved caspase-3, phospho-S6, and hexokinase II. Digital slides were analyzed with the Definiens’ Tissue Studio (Definiens Inc. Parsippany, NJ) to determine adenocarcinoma tumor area and necrotic tumor area.

Momcilovic M., McMickle R., Abt E., Seki A., Simko S.A., Magyar C., Stout D.B., Fishbein M.C., Walser T.C., Dubinett S.M, & Shackelford D.B. (2015). Heightening energetic stress selectively targets LKB1-deficient non-small cell lung cancers. Cancer research, 75(22), 4910-4922.

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