Total proteins from cell lines and tissues were extracted with RIPA buffer and then quantified by BCA analysis. Subsequently, 20 µg of total protein per sample (10 µL per lane) was separated using sodium lauryl sulphate–polyacrylamide gel electrophoresis (10% polyacrylamide gel) before the proteins were transferred to a PVDF membrane. After incubation with primary antibodies overnight, the membranes were then incubated with secondary antibody. Finally, target protein bands were detected using a chemiluminescence system. The antibodies used targeted the following proteins: AKT (Cell Signaling #9272s), Caspase-9 (Proteintech 10380-1-AP), Caspase-3 (Proteintech 66470-2-Ig), Caspase-8 (Proteintech 66093-1-Ig), p-ATK (Cell Signaling #4060), BAX (Proteintech 60267-1-Ig), Vimentin (Proteintech 10366-1-AP), N-cadherin (Proteintech 22018-1-AP), E-cadherin (Proteintech 20874-1-AP), GAPDH (Signalway Antibody #21612) and METTL3 (ABclonal A8370).
Caspase 8
Manufactured by Proteintech
Sourced in United States, China
Caspase-8 is an enzyme that plays a crucial role in the initiation of the apoptosis (programmed cell death) pathway. It acts as an initiator caspase, activating downstream effector caspases to execute the cell death process.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3, by Proteintech (16 mentions)
Bcl 2, by Proteintech (12 mentions)
Caspase 9, by Proteintech (11 mentions)
Bcl2 associated x (bax), by Proteintech (9 mentions)
β actin, by Proteintech (7 mentions)
E cadherin, by Proteintech (4 mentions)
N cadherin, by Proteintech (4 mentions)
Vimentin, by Proteintech (4 mentions)
Gapdh, by Proteintech (4 mentions)
Parp1, by Proteintech (4 mentions)
Cyclin d1, by Proteintech (4 mentions)
P atk, by Cell Signaling Technology (3 mentions)
Gapdh, by Signalway Antibody (3 mentions)
Cleaved caspase 3, by Proteintech (3 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Caspase 7, by Proteintech (2 mentions)
Bcl2 associated x (bax), by Abcam (2 mentions)
Gsdme, by Proteintech (2 mentions)
Rabbit polyclonal antibodies against human caspase 3, by Proteintech (2 mentions)
37 protocols using caspase 8
1
Western Blot Analysis of Protein Markers
2
Assessing Apoptosis Markers in MSCs
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MSCs were treated with 20 ng/mL TNF-α and 10 µg/mL CHX at different time points (6 h, 12 h and 24 h). For comparison of anti-apoptosis effect of MSC, EGFP-MSC and sTNFRII-MSC, cells were treated with 20 ng/mL TNF-α and 10 µg/mL CHX for 12 h. Cell total protein was harvested after lysing and centrifuging at 12,000 × g for 15 min at 4 ℃. Supernatant was collected and added with 5 × protein loading buffer (Beyotime Biotechnology, China), then the protein samples were denatured at 100 ℃ for 10 min. The denatured protein samples were separated by polyacrylamide gel electrophoresis and transferred electrophoretic ally to a polyvinylidene fluoride membrane (Millipore, MA, USA). The dilution of primary antibody of Bcl-2, Bax, Caspase 3, Cleaved Caspase 3, Caspase 8, Cleaved Caspase 8, LC3B I/II, TRIB3 and β-actin (all from Proteintech, USA) is 1:800. The dilution of second antibody of goat anti-rabbit/mouse is 1:10,000 (ZSGB-BIO, Beijing, China). The membranes were scanned with an ImageQuant LAS 4000mini (GE Healthcare) and the density of protein bands was analyzed by ImageJ software (National Institutes of Health).
3
Tianma Gouteng Decoction for Cardiovascular Health
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Tianma Gouteng Decoction from Gastrodia elata 10 g, Uncaria 15 g (later fried), abalone shell 20 g (first fried), Gardenia 6 g, Scutellaria baicalensis Georgi 6 g, achyranthes root 12 g, Eucommia ulmoides Oliv. 12 g, Leonurus japonicus 12 g, Loranthus parasiticus 12 g, dried caulis of polygoni multiflori 12 g, and Poria cocos 12 g. All of the above ingredients were provided by the Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. TGD is concentrated into granules; each 1 g granule is equal to 10 g of raw materials and complies with the guidelines of Good Manufacturing Practices and Good Laboratory Practices formulated by Chinese government agencies. The TGD was dissolved in pure water to prepare solutions having a concentration of 200 mg/mL or 400 mg/mL for the experiment.
Saline was purchased from China Otsuka Pharmaceutical Co., Ltd. (Tianjin, China). The enzyme-linked immunosorbent assay (ELISA) kits, including prostacyclin (PGI2), thromboxane A2 (TXA2), angiotensin II (Ang II), and endothelin 1 (EDN1), were all obtained from Uscn Life Science, Inc. (Wuhan, China). The primary antibodies of GAPDH, AKT, p-AKT, caspase 8, caspase 7, caspase 3, and secondary antibodies were purchased from Proteintech Inc. (IL, USA). The antibodies of OPG, TRAIL, and DR5 were purchased from Abcam PLC (Cambridge, UK).
Saline was purchased from China Otsuka Pharmaceutical Co., Ltd. (Tianjin, China). The enzyme-linked immunosorbent assay (ELISA) kits, including prostacyclin (PGI2), thromboxane A2 (TXA2), angiotensin II (Ang II), and endothelin 1 (EDN1), were all obtained from Uscn Life Science, Inc. (Wuhan, China). The primary antibodies of GAPDH, AKT, p-AKT, caspase 8, caspase 7, caspase 3, and secondary antibodies were purchased from Proteintech Inc. (IL, USA). The antibodies of OPG, TRAIL, and DR5 were purchased from Abcam PLC (Cambridge, UK).
4
Western Blot Analysis of Mouse Heart Proteins
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Mouse heart tissue/cells were lysed for 30 min with RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Beyotime, China) on ice. The lysate was centrifuged at 12 000 rpm for 15 min at 4 °C, and the protein concentrations of the supernatant were analyzed with a BCA kit (Beyotime, China). Each sample (20 µg of protein) was separated by SDS‒PAGE gels and transferred to PVDF membranes. The membranes were immersed in 5% milk in TBST buffer for 1 h at room temperature, followed by incubation with primary antibodies against β‐actin (CST, 1:1000), bax (Abcam, 1:1000), caspase3 (CST, 1:1000), caspase 8 (CST, 1:1000), Cyt‐c (Proteintech, 1:1000), GPx‐4 (Abcam, 1:1000), Bcl‐2 (Affinity, 1:1000), TGF‐β (CST, 1:1000), COX‐2 (Affinity, 1:1000), VEGF A (CST, 1:1000), FAK (CST, 1:1000), ERK1/2 (CST, 1:1000), p38 (CST, 1:1000), transferrin receptor (Abcam, 1:1000), and ferroportin (Novus, 1:1000) at 4 °C overnight. Then, the membranes were washed three times with TBST, followed by incubation with secondary antibodies (1:10 000) for 1 h at room temperature. The bands were visualized by using a gel documentation system (Bio‐Rad, USA) and quantified by ImageJ software.
5
Necroptosis Pathway Protein Analysis
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Caspase-3, Caspase-8, RIPK1, RIPK3 (rabbit antibody), MLKL, OGT and OGA antibodies were purchased from Proteintech (Wuhan, China). Phospho-RIPK1, phospho-RIPK3, phospho-MLKL and secondary antibodies were from Cell Signaling Technology (Beverly, MA, USA). MPO antibody was provided by ABclonal Technology (Wuhan, China). CD68 antibody was from Abcam (Cambridge, MA). O-GlcNAc antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Another RIPK3 (mouse) antibody was purchased from Novus Biologicals (Littleton, CO, USA). 2,4,6-trinitrobenzene sulfonic acid (TNBS) was from Thermo Fisher Scientific (CA, USA). Protein A/G magnetic beads were purchased form Bimake (Shanghai, China). The TUNEL staining and immunohistochemistry kits were purchased from Wuhan Gugeshengwu Technology Co.,Ltd. (Wuhan, China). All other regular reagents were from Wuhan Gugeshengwu Technology Co.,Ltd. unless otherwise specified.
6
Protein Expression Analysis by Western Blot
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Protein samples were extracted with lysis buffer. 15 μg protein samples from each group were loaded onto the SDS polyacrylamide gel for electrophoresis and transferred to NC membranes (Millipore, Billerica, MA, USA). After blocking in 5% non-fat milk at room temperature for 2 h, primary antibodies: NPRA (1:1000; Santa Cruz Biotechnology), NPRC (1:1000; Santa Cruz Biotechnology), PGRMC1 (1:1000; Cell Signaling), PGRMC2 (1:1000), Bax (1:2000; Proteintech), Bcl-2 (1:2000; Proteintech), Caspase 8 (1:1000; Proteintech), Caspase 9 (1:1000; Proteintech), PCNA (1:2000; Proteintech), EGFR (1:500; Proteintech), p-EGFR (1:500; Cell Signaling), ERK 1/2 (1:500; Beyotime Biotechnology), p-ERK 1/2 (1:500; Beyotime Biotechnology), p-c-Fos (1:500; Cell Signaling), c-Fos (1:500; Cell Signaling), p-c-Jun (1:500; Cell Signaling), c-Jun (1:500; Cell Signaling) and GAPDH (1:2000; Proteintech) were incubated at 4 °C overnight. Next day, secondary antibodies (1:2000; Cell Signaling) were incubated for 45 min at room temperature. ECL detection system was used to visualize the bands. All experiments were repeated at least three separate times.
7
Apoptosis Signaling Pathway Analysis
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Total protein was extracted from tissues after using RIPA lysis and centrifugation, and was boiled at 95℃ for 10 min. Protein was transferred to the PVDF membrane after electrophoretic separation. The membrane was then incubated with the diluted primary antibodies: Cleaved Caspase 3 (Proteintech, 25128-1-AP, 1:1000), Caspase 3 (Proteintech, 19677-1-AP, 1:10000), Caspase 8 (Proteintech, 13423-1-AP, 1:800), Cleaved Caspase 8 (CST, 8592 S, 1:1000), TRAIL (CST, 3219 S, 1:1000) overnight at 4℃ and 1:10000 secondary antibodies (Proteintech, SA00001-1/, SA00001-2, 1:10000) for 1 h the next day. Chemiluminescence was performed for the results.
8
Detecting Apoptosis Pathway Proteins
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Rabbit polyclonal antibodies against human caspase-3, caspase-8, caspase-9, and GSDME were purchased from Proteintech (Rosemont, IL, USA). The ElivisionTM Plus Kit and DAB color developing kit were purchased from Fuzhou Maixin Biological Company (China).
9
Molecular Mechanisms of Ghrelin-Induced Autophagy
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Ghrelin was purchased from ProSpec (Ness-Ziona, Israel). Con A was purchased from Sigma-Aldrich (St Louis, MO, USA). Perifosine was provided by Keryx Biopharmaceuticals (New York, NY, USA). Antibodies against IL-1β, IL-6, TNF-α, total Akt, and p-Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LC3, Beclin 1, Bcl-2, and Bax were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against caspase 3, caspase 8, and caspase 9 were purchased from Proteintech (Chicago, IL, USA).
10
Paraffin-Embedded Tissue Section Analysis
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Samples containing tissues from each group were fixed in 10% buffered formalin for 24 h at 4°C, embedded into paraffin and sliced into 4–5 µm-thick sections, The wax sheets were smoothed on polylysine-treated slides in the spreading machine tank (45°C). The surrounding water was drained with absorbent paper and tissues were place in an oven at 60°C for 4 h. Subsequently, H&E staining was performed (hematoxylin staining for 5–10 min; 0.6% ammonia reflux blue; eosin staining for 3–5 min; treatment with 70% ethanol and 80% ethanol for 10–20 sec, 95% ethanol for 3–5 min, 100% ethanol for 3–5 min, and transparent xylene for 3–5 min. All the operations were performed at room temperature) and tissues were observed using a light microscope (magnification, ×200; Olympus Corporation) to compare the pathological features of samples. IHC was performed using caspase-3 (1:250; cat. no. 19677-1-AP; ProteinTech Group, Inc.), caspase-8 (1:250; cat. no. 13426-1-P; ProteinTech Group, Inc.), Bax (1:2,500; cat. no. 50599-2-lg; ProteinTech Group, Inc.) and Bcl-2 (1:1,000; cat. no. 12789-1-AP; ProteinTech Group, Inc.) antibodies (incubated at 4°C overnight; reheated at room temperature for 45 min), and secondary antibody (1:1,000; Servicebio, Inc.; cat. no. GB24303; 37°C, 1 h) antibodies.
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