Caspase 12

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Caspase-12 is a member of the caspase family of enzymes. It is involved in the process of apoptosis, or programmed cell death. Caspase-12 plays a role in the endoplasmic reticulum (ER) stress-induced apoptotic pathway. Its primary function is to initiate and execute the apoptotic process in response to certain cellular stressors.

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Anti-caspase-12 (5 citations) Cleaved-caspase-12 (2 citations)

17 protocols using caspase 12

Bufalin-Induced Apoptosis in Leukemia Cells

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Cell lines K562 and K562/A02 cells were purchased from the Shanghai Institute of Cell Sciences, Chinese Academy of Sciences (Shanghai, PRC). ADM and bufalin were purchased from Sigma-Aldrich (St. Louis, MO). Nrf2, HO-1, P-gp, Bax, Bcl-2, cyctc, PAPR, IRE1α, TRAF2, JNK, p-JNK, and Caspase-12 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). The apoptosis detection kit was purchased from KeyGen Biotech (Nanjing, PR China). RT-PCR and HiPerfect transfection kits were purchased from Qiagen (Hilden, Germany).

Xie Y., Yan X, & Sun L. (2019). The Mechanism of Bufalin-Induced Apoptosis of K562/A02. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 25, 2542-2552.

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Western Blot Analysis of Protein Markers

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Total protein was extracted from heart tissues and cell samples using ice‐cold RIPA lysis buffer (Cell Signaling Technology). Denatured protein samples were loaded (30 μg for each well) onto 12% sodium dodecylsulfate‐polyacrylamide electrophoretic gels and separated, and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The PVDF membranes were immunoblotted with primary antibodies against Nesfatin‐1 (1:500, R&D Systems), β‐actin (1:5000, Santa Cruz Biotechnology), p‐Akt (1:2000, Abcam), Akt (1:2000, Abcam), p‐ERK (1:1000, Santa Cruz Biotechnology), ERK (1:1000, Santa Cruz Biotechnology), CHOP (1:500, Cell Signaling Technology), ATF6 (1:500, Proteintech), GPR78 (1:1000, Novus Biologicals) and caspase‐12 (1:1000, Santa Cruz Biotechnology), respectively. Immunoreactivity was determined with enhanced chemiluminescence (ECL) method and quantified by Image J software (version 1.47, NIH, Bethesda, MD, USA).

Su R., Geng X., Yang Y, & Yin H. (2021). Nesfatin‐1 inhibits myocardial ischaemia/reperfusion injury through activating Akt/ERK pathway‐dependent attenuation of endoplasmic reticulum stress. Journal of Cellular and Molecular Medicine, 25(11), 5050-5059.

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Oxidative Stress and Signaling Pathways in Cell Model

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GP-17 (purity > 98%) was obtained from Shanghai Winherb Medical S&T Development (Shanghai, China). The cell culture materials, including fetal bovine serum, Dulbecco's modified Eagle's medium, and penicillin/streptomycin, were obtained from Gibco (NY, USA). The compounds used in Krebs-Henseleit (KH) buffer were purchased from Sigma-Aldrich Chemicals. The kits of malondialdehyde (MDA), catalase (CAT), lactate dehydrogenase (LDH), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). The primary antibodies, PI3K, P-PI3K, AKT, P-AKT, P38, P–P38, GRP78, P-PERK, PERK, P-eIf2α, eIf2α, ATF6, IRE1, P-JNK, JNK, CHOP, caspase-12, BAX, Bcl-2, BAD, and GAPDH were supplied by Santa Cruz Biotechnology (CA, USA).

Yu Y., Wang M., Chen R., Sun X., Sun G, & Sun X. (2019). Gypenoside XVII protects against myocardial ischemia and reperfusion injury by inhibiting ER stress–induced mitochondrial injury. Journal of Ginseng Research, 45(6), 642-653.

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Investigating Cellular Stress Mechanisms

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4-phenylbutiric acid (4-PBA) and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO). The kits for determining lactate dehydrogenase (LDH), total reactive oxygen species (ROS), malondialdehyde (MDA) contents, total creatine kinase (CK), superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) were obtained from Jiancheng Bioengineering Institute (Nanjing, China). In Situ Cell Death Detection Kits (Fluorescein) were purchased from Roche (Basel, Switzerland). Primary antibodies against Grp78, p-PERK, PERK, CHOP, Caspase-12, JNK, p-JNK, Bax, Bcl-2, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Jian L., Lu Y., Lu S, & Lu C. (2016). Chemical Chaperone 4-Phenylbutyric Acid Reduces Cardiac Ischemia/Reperfusion Injury by Alleviating Endoplasmic Reticulum Stress and Oxidative Stress. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 5218-5227.

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Protein Expression Profiling in Cardiomyocytes

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Cardiomyocytes were lysed, and protein extraction was performed as previously described.[12 (link)] The soluble supernatant of the extracts was determined by the Bradford method, and 16 samples of 50 mg protein were prepared and separated on 10% acrylamide gels. The separated proteins were electrophoretically transferred to nitrocellulose membranes, blocked with 5% bovine serum albumin in tris-buffered saline Tween 20, containing 20 mmol/l tris-HCl (pH 7.6), 137 mmol/l NaCl and 0.1% Tween 20. The membranes were incubated with the antibodies against CRT (1:1000, Stressgen), Nrf2 (1:1000, Santa Cruz), ATF4 (1:1000, Santa Cruz), glucose-regulated protein 78 (GRP78) (1:1000, Santa Cruz), CHOP (1:200, Santa Cruz), caspase-12 (1:1000, Santa Cruz), caspase-3 (1:1000, Santa Cruz), Bax (1:200, Santa Cruz), Bcl-2 (1:200, Santa Cruz) and GAPDH (1:500, Santa Cruz), respectively overnight at 4°C. After incubation for 2 hours with horseradish peroxidase-conjugated secondary antibodies, the reaction was visualized using an enhanced chemiluminescence kit (Santa Cruz Biotechnology). The integrated optical density (mean intensity × area) of proteins was quantified using Image-Pro Plus. The relative level of analyzed protein expression was normalized to that of GAPDH.

Xu F.F, & Liu X.H. (2015). Calreticulin Translocation Aggravates Endoplasmic Reticulum Stress-associated Apoptosis during Cardiomyocyte Hypoxia/Reoxygenation. Chinese Medical Journal, 128(3), 353-360.

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Evaluating Apoptosis Pathway Proteins

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Cells were lysed in PRO-PREP solution (Intron, Daejeon, Korea). Total protein was measured using BCA protein assay kit (Thermo Scientific, Rockford, IL). Samples were run on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies. Blots were then incubated with peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (Intron, Daejeon, Korea). The following primary antibodies were used: poly(ADP-ribose) polymerase-1 (PARP-1), activating transcription factor-4 (ATF-4), caspase-4, caspase-12, β-catenin, and α-tubulin (Santa Cruz Biotechnologies, Santa Cruz, CA); CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase-3 (Cell Signaling Technology, Beverly, MA); and β-actin (Sigma-Aldrich, St. Louis, MO).

Zhang Q.L., Lian D.D., Zhu M.J., Li X.M., Lee J.K., Yoon T.J., Lee J.H., Jiang R.H, & Kim C.D. (2019). Antitumor Effect of Albendazole on Cutaneous Squamous Cell Carcinoma (SCC) Cells. BioMed Research International, 2019, 3689517.

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Protein Fractionation and Western Blot Analysis

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Tissues or cells were homogenized in RIPA lysis buffer containing a protease
inhibitor cocktail (Roche). Membrane protein, cytosolic protein and
mitochondrial protein were isolated using the Membrane/Cytosol or
Mitochondria/Cytosol Protein Fractionation Kit according to the
manufacturer’s protocol (Beyotime Inst Biotech). Identical amounts
of protein samples were separated by SDS-PAGE, blotted onto a PVDF membrane, and
incubated with antibodies against p47phox (Bioworld), Mn-SOD (ABclonal), Bax,
Bcl-2, caspase-12, CHOP, p-JNK, β-actin (Santa Cruz), p53,
caspase-3, cleaved caspase-3, GRP78 (Cell Signaling Technology), JNK (ABclonal),
p-p53, or cytochrome-c (Abcam) at 4 °C overnight. Then,
goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz) were added, and the
blots were developed with an ECL plus kit (Amersham Biosciences). The images
were scanned with an Epson scanning system, and the data were analyzed with
Quantity-one software. The data are expressed as the values relative to the sham
or control value.

Zhong D., Wang H., Liu M., Li X., Huang M., Zhou H., Lin S., Lin Z, & Yang B. (2015). Ganoderma lucidum polysaccharide peptide prevents renal ischemia reperfusion injury via counteracting oxidative stress. Scientific Reports, 5, 16910.

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Endoplasmic Reticulum Stress and Inflammation

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EP, thapsigargin (THA, an ERS inducer), tumor necrosis factor-α (TNF-α), dimethyl sulfoxide (DMSO) and the antibody against TNF-α were purchased from the Sigma-Aldrich Company (St. Louis, MO, USA). The Cell Counting Kit-8 (CCK8) was purchased from Dojindo (Kumamoto, Japan). PERK siRNA and the antibodies against GRP78, intercellular cell adhesion molecule 1 (ICAM-1), matrix metalloproteinase 9 (MMP9) and caspase12 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human soluble adhesion molecule ICAM-1 (sICAM-1), soluble E-selectin (sE-selectin), interleukin (IL-8) and monocyte chemoattractant protein-1 (MCP-1) ELISA kits were obtained from R&D Systems (Minneapolis, MN, USA). The antibodies against phosphorylated-PERK (p-PERK), PERK, ATF4 and β-actin were obtained from Cell Signaling Technology (Beverly, MA, USA). The rabbit anti-goat, goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from the Zhongshan Company (Beijing, China).

Wang G., Liu K., Li Y., Yi W., Yang Y., Zhao D., Fan C., Yang H., Geng T., Xing J., Zhang Y., Tan S, & Yi D. (2014). Endoplasmic Reticulum Stress Mediates the Anti-Inflammatory Effect of Ethyl Pyruvate in Endothelial Cells. PLoS ONE, 9(12), e113983.

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Notoginsenoside R1 Modulates ER Stress

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Notoginsenoside R1 (NGR1, CID: 441934, molecular weight = 933.15; purity > 98%) was supplied by Shanghai Winherb Medical S&T Development (Shanghai, China). Tunicamycin (TM) from Streptomyces was purchased from Sigma (St. Louis, MO, USA), and the 4-phenylbutyric acid (4-PBA, CAS.NO. 1821-12-1) was purchased from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). All cell culture materials, Dulbecco’s modified Eagle’s medium (DMEM), foetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco (NY, USA). The kits for determining the malondialdehyde (MDA) content and theactivity of creatine kinase (CK), catalase (CAT), lactate dehydrogenase (LDH), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were obtained from Jiancheng Bioengineering Institute (Nanjing, China). Primary antibodies against JNK, P-JNK, CHOP, GRP78, ATF6, P-PERK, PERK, IRE1, eIf2α, P-eIf2α, Caspase-12, Bcl-2, BAX, BAD and β-actin were obtained from Santa Cruz Biotechnology (CA, USA).

Yu Y., Sun G., Luo Y., Wang M., Chen R., Zhang J., Ai Q., Xing N, & Sun X. (2016). Cardioprotective effects of Notoginsenoside R1 against ischemia/reperfusion injuries by regulating oxidative stress- and endoplasmic reticulum stress- related signaling pathways. Scientific Reports, 6, 21730.

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Western Blot Analysis of Apoptosis Markers

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Western blot analysis were performed as described previously.¹⁴ (link) Antibodies were as follows: cleaved caspase-3, cleaved caspase-7, caspase-7, PARP, Beclin1, ATG5, ATG12, LC3B (Cell Signaling, Danvers, MA, USA), and Bcl-2, p-IRE1α, p-PERK, ATF6, Bip, CHOP, caspase-12, and actin (Santa Cruz Biotechnology, CA, USA). Finally, the membranes were visualized with the enhanced chemiluminescence (ECL) kit (Amersham, Cardiff, UK) using the LAS-3000 Image Reader of Luminescence Image Analyzer (FUJIFILM Life Science, Tokyo, Japan). The protein levels were quantified using Multi gauge V3.0 software program (FUJIFILM Life Science) and were normalized to the expression of the actin levels.

Yoon H.E., Ahn M.Y., Kim Y.C, & Yoon J.H. (2022). Involvement of endoplasmic reticulum stress and cell death by synthesized Pa-PDT in oral squamous cell carcinoma cells. Journal of Dental Sciences, 17(4), 1722-1730.

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