Cle caspase 3

Manufactured by Cell Signaling Technology

Sourced in United States

Cle-Caspase-3 is a laboratory product developed by Cell Signaling Technology. It is a recombinant, active human caspase-3 enzyme. Caspase-3 is a protease that plays a central role in the execution of the apoptotic program.

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Caspase-3 (2138 citations) Caspases (2 citations)

4 protocols using cle caspase 3

Multimodal Immunofluorescence Staining Protocol

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The coronal sections (25 µm) were incubated with 10% normal donkey serum for 1 h at room temperature in PBS containing 0.1% Triton X-100, followed by incubation with appropriate primary antibodies overnight at 4°C in the same buffer. The following primary antibodies were used in different combinations: anti-NeuN (1:500, Millipore, MA), Iba1, GFAP and PARP1 (1:200, Proteintech, IL), Caspase-1 and Cle-Caspase-1 (1:50, Santa Cruz Biotechnology), Cle-caspase-3 and Cle-caspase-9 (1:50, Cell Signaling Technology). After primary antibody incubation, the sections were washed 4 times for 10 min at room temperature, followed by incubation with the appropriate combination of Alexa Fluor donkey anti-mouse/rabbit/goat secondary antibody (1:500, Thermo Fisher Scientific) for 1 h at room temperature. For negative control staining, the primary antibody was omitted during immunostaining. The sections were then washed, mounted and coverslipped in Vectashield mounting medium (H-1200, Vector Laboratories). Three to five sections from each animal (200 µm apart) were selected for confocal microscopy. The captured images were processed and analyzed using LSM510 Meta imaging software.

Zhao N., Zhuo X., Lu Y., Dong Y., Ahmed M.E., Tucker D., Scott E.L, & Zhang Q. (2016). Intranasal Delivery of a Caspase-1 Inhibitor in the Treatment of Global Cerebral Ischemia. Molecular neurobiology, 54(7), 4936-4952.

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Protein Extraction and Western Blot Analysis

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Protein extracts from the heart and liver tissue were obtained by homogenization in lysis buffer (100 mg/mL) as mentioned previously [1 (link), 26 (link)]. The protein concentrations were determined by Lowry protein assay and the samples were electrophoresed in a 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were then transferred to PVDF membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The membranes were blocked using 5% non-fat milk for 1 h. Monoclonal primary antibodies were diluted 1:1000 in antibody binding buffer (TBS) and used for hybridization overnight (4 °C) with the following antibodies ANP, phosphor-Akt, β-Actin, Bax, BNP, Cytochrome C, phosphor-GATA4, PGC1α (sc-20158, sc-7985, sc-47778, sc-526, sc-18818, sc-13560, sc-32823-R, sc-13067, Santa Cruz); phosphor-AMPK, Cle-Caspase-3, phosphor-FOXO3a, Sirt1 (#2535, #9664, #9466, #9475s, Cell Signaling, The Netherlands). Following hybridization with appropriate secondary antibodies the membranes were washed in for 10 min thrice. The blots were detected in chemiluminescent detection using ECL with Fujifilm LAS-3000 (GE Healthcare Life Sciences).

Asokan S.M., Wang T., Wang M.F, & Lin W.T. (2020). A novel dipeptide from potato protein hydrolysate augments the effects of exercise training against high-fat diet-induced damages in senescence-accelerated mouse-prone 8 by boosting pAMPK / SIRT1/ PGC-1α/ pFOXO3 pathway. Aging (Albany NY), 12(8), 7334-7349.

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Immunohistochemical Analysis of Tumor Proliferation and Apoptosis

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Tumor tissues were fixed, dehydrated, embedded in paraffin, and serially sectioned at a thickness of 4 μm. Then the sections were incubated with primary and secondary antibodies. Briefly, the paraffin‐embedded tissue was dried, dewaxed, and rehydrated. Then the sections were rinsed phosphate with buffered saline solution (PBS, Beyotime, Shanghai, China) and blocked with bovine serum albumin (Beyotime, Shanghai, China). The sections were incubated with Ki-67 monoclonal antibody (34,330, Cell Signaling Technology, MA, USA) and cle-caspase3 (9664, Cell Signaling Technology, MA, USA) at 4 °C overnight. The sections were incubated with a secondary antibody the next day for 30 min at 37 °C. The diaminobenzene was used as the chromogen, and hematoxylin was used as the nuclear counterstain. The IHC scores were obtained by multiplying the percentage of positive tumor cells and the intensity of staining. The value of positive tumor cells was defined as 0 (no staining), 1 (0%–25%), 2 (25%–50%) and 3 (> 50%). The value of intensity of staining was defined as 0, 1, 2 and 3^{25 (link)}.

Huang Y., Yang W., Yang L., Wang T., Li C., Yu J., Zhang P., Yin Y., Li R, & Tao K. (2023). Nrf2 inhibition increases sensitivity to chemotherapy of colorectal cancer by promoting ferroptosis and pyroptosis. Scientific Reports, 13, 14359.

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Western Blot Analysis of Apoptosis Signaling

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Cells that needed examination were lysed in RIPA buffer to extract the total cellular protein. Protein concentration was determined and 60 µg of each protein sample was boiled at 100°C in SDS sample buffer for 10 min, electrophoresed on 10% SDS/PAGE gels, and then transferred to polyvinylidene difluoride (PVDF) membranes. Following protein transfer, the membranes were blocked by 5% skimmed milk proteins for 90 min, followed by incubation at 4°C overnight with the primary antibodies. Membranes were incubated with correlate secondary antibody at room temperature for 1 h on the second day, and specific protein bands were detected with an enhanced chemiluminescence (ECL) assay kit (BD, USA). The primary antibodies used were as follows: Cle-PARP, Bcl-2, Bax, p-IKK, IKK, IκB-α, and GAPDH (Santa Cruz Biotechnology, USA) and Cle-caspase-3 (Cell Signaling Technology, USA).

Chen J., Zhang L., Shu Y., Chen L., Zhu M., Yao S., Wang J., Wu J., Liang G., Wu H, & Li W. (2017). Curcumin Analogue CA15 Exhibits Anticancer Effects on HEp-2 Cells via Targeting NF-κB. BioMed Research International, 2017, 4751260.

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