Anti cleaved caspase 3 d175

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-cleaved-Caspase 3 (D175) is a laboratory reagent used to detect the presence of cleaved Caspase 3, a protein involved in the apoptosis (programmed cell death) process. This primary antibody specifically recognizes the cleaved form of Caspase 3, which is a hallmark of cells undergoing apoptosis.

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Cleaved caspase-3 (3596 citations) Caspase-3 (2138 citations) Anti-cleaved caspase-3 (1212 citations) Anti-caspase-3 (744 citations) Rabbit anti-cleaved caspase-3 (D175) (4 citations) Caspases (2 citations)

5 protocols using anti cleaved caspase 3 d175

Western Blot Analysis of Protein Levels

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Standard western blot assays were used to analyze the levels of protein [14 , 37 (link)]. Anti-p53 (FL393, 1:2 000 dilution, Santa Cruz), anti-MDM2 (2A10, 1:1 000), anti-Flag (F7425,1:10 000 dilution, Sigma), anti-BAG5(ARP61996-P050, 1:1 000 dilution, Aviva Systems Biology, San Diego, CA, USA), anti-HA (sc-7392, 1:2 000 dilution, Santa Cruz), anti-CHIP (sc-66830, 1:1 000 dilution, Santa Cruz), anti-cleaved-Caspase 3 (D175, 1:1 000 dilution, Cell Signaling, Danvers, MA, USA) and anti-β-actin(A5316, 1:20 000 dilution, Sigma) antibodies were used to determine the levels of p53, MDM2, Flag-BAG5, BAG5, HA-BAG5, CHIP, cleaved caspase 3 and β-actin, respectively.

Yue X., Zhao Y., Huang G., Li J., Zhu J., Feng Z, & Hu W. (2016). A novel mutant p53 binding partner BAG5 stabilizes mutant p53 and promotes mutant p53 GOFs in tumorigenesis. Cell Discovery, 2, 16039-.

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Cytotoxicity Evaluation of Anti-Cancer Agents

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The PARPi olaparib (AZD2281) was purchased from Selleckchem (Houston, TX, USA). Doxorubicin was purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents, including carboplatin (Dong-A ST, Seoul, Korea), cisplatin (JW Pharmaceutical, Seoul, Korea), and paclitaxel (Samyang Biopharm, Gyeonggi-do, Korea) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). MTT was purchased from Amresco (Solon, OH, USA). Primary antibodies used in this study included the following; anti-cleaved caspase 3 (D175, #9661; Cell Signaling Technology; Danvers, MA, USA), phospho-histone H2AX (Ser 139, sc-517348), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sc-47724; Santa Cruz Biotechnology; Dallas, TX, USA), and anti-β-actin (A5441; Sigma-Aldrich) antibodies.

Ryu H., Kim H.J., Song J.Y., Hwang S.G., Kim J.S., Kim J., Bui T.H., Choi H.K, & Ahn J. (2019). A Small Compound KJ-28d Enhances the Sensitivity of Non-Small Cell Lung Cancer to Radio- and Chemotherapy. International Journal of Molecular Sciences, 20(23), 6026.

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Protein Extraction and Western Blot Analysis

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The cellular proteins were solubilized in a Tris buffer containing 2% sodium dodecyl sulfate and 10% glycerol. The following antibodies were used for Western blotting: anti-SPP1 (AF1433; R&D Systems), anti-CD204 (clone SRA-E5; CosmoBio), anti-Bcl-2 (D17C4; Cell Signaling Technology, Danvers, MA, USA), anti-Bax (D2E11; Cell Signaling Technology), anti-cleaved caspase-3 (D175; Cell Signaling Technology), anti-cleaved PARP (Asp214; D64E10; Cell Signaling Technology), anti-Bmi1 (EPR3745(2); abcam, Cambridge, UK), and β-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA). The detected bands were quantitatively measured using Image J [28 (link)]. The original WB can be found in the Supplementary Material File S1.

Matsubara E., Komohara Y., Esumi S., Shinchi Y., Ishizuka S., Mito R., Pan C., Yano H., Kobayashi D., Fujiwara Y., Ikeda K., Sakagami T, & Suzuki M. (2022). SPP1 Derived from Macrophages Is Associated with a Worse Clinical Course and Chemo-Resistance in Lung Adenocarcinoma. Cancers, 14(18), 4374.

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Quantification of Neuronal Protein Expression

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The concentrations of total protein extracted from primary cultured cortical neurons or tissues of brain were quantified using BCA (Bicinchoninic acid assay). Then, protein was electrophoresized with SDS‐PAGE gel (12%), further transferred onto polyvinylidene difluoride membranes. TBS containing nonfat milk was used to block the membranes, then it was incubated with SIRT1 antibodies (1:800; Cell Signaling Technology, Inc., Danvers, MA, USA), QKI 6 (1:2000; Abcam, Cambridge, UK), PGC‐1α (1:1000; Abcam, Cambridge, UK), PPARγ(1:1500; Abcam, Cambridge, UK), or anti‐cleaved caspase‐3 (D175; 1:1200; Cell Signaling Technology, Beverly, MA), and β‐actin mouse mAb (8H10D10; CST #3700; 1:1000; Cell Signaling Technology, Beverly, MA), respectively. Then, those were further incubated using the HRP (horseradish peroxidase)‐conjugated second antibodies (1:1000 dilution; Cell Signaling Technology). In the end, the image of blots was obtained using the electrochemiluminescence detection system.

Liu R., Li H., Deng J., Wu Q., Liao C., Xiao Q, & Chang Q. (2021). QKI 6 ameliorates CIRI through promoting synthesis of triglyceride in neuron and inhibiting neuronal apoptosis associated with SIRT1‐PPARγ‐PGC‐1α axis. Brain and Behavior, 11(8), e2271.

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Immunostaining of Stem Cell Markers

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Primary antibodies were used at the following concentrations for immunostaining: 2.5μg/mL anti-Activin A (R&D Systems; MAB3381), 0.75μg/mL anti-pSMAD2 (Ser465/467) (Cell Signaling; 3108T), 2.5μg/mL anti-EpCAM/CD326 (BioLegend; 118201), 1μg/mL anti-Ki-67 (Invitrogen; PA5-19462), 7μg/mL anti-Ac-alpha-Tubulin (K40), 0.16μg/mL anti-Cleaved Caspase 3 (D175) (Cell Signaling; 5335S), 0.2μg/mL anti-NCAM1 (Cell Signaling; 99746T), 1μg/mL anti-SOX9 (Millipore; AB5535), 2.5μg/mL anti-EpCAM/CD326-647 (BioLegend; 118212). Secondary antibodies used for immunostaining: 4μg/mL donkey anti-rabbit Alexa Fluor 555 (ThermoFisher; A31572), 4μg/mL donkey anti-rat Alexa Fluor 488 (ThermoFisher; A21208), 4μg/mL donkey anti-mouse Alexa Fluor 555 (ThermoFisher; A31570), and 5μg/mL goat anti-rabbit HRP (ThermoFisher; G21234).

Hrncir H.R., Bombin S., Goodloe B., Hogan C.B., Jadi O, & Gracz A.D. (2024). Sox9 links biliary maturation to branching morphogenesis. bioRxiv.

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