Ab87540

Manufactured by Abcam

Sourced in United States

Ab87540 is a laboratory equipment product. It is a device used for the detection and measurement of specific molecules or compounds in a sample.

Automatically generated - may contain errors

Lab products found in correlation

Ab32529, by Abcam (4 mentions) Ab84400, by Abcam (2 mentions) Ab32539, by Abcam (2 mentions) Bs7070, by Bioworld Technology (2 mentions) Bs1511, by Bioworld Technology (2 mentions) Ab91109, by Abcam (2 mentions) Epics xl, by Beckman Coulter (2 mentions) Anti rat cd45 pe, by Thermo Fisher Scientific (2 mentions) Anti rat cd8a apc, by Thermo Fisher Scientific (2 mentions) Anti rat cd44 pe, by Thermo Fisher Scientific (2 mentions) Anti rat cd62l, by BioLegend (2 mentions) Ab109012, by Abcam (1 mentions) Rapamycin, by Selleck Chemicals (1 mentions) 3 methyladenine 3 ma, by Selleck Chemicals (1 mentions) Ab8227, by Abcam (1 mentions) Ab2413, by Abcam (1 mentions) Ab34710, by Abcam (1 mentions) Ab2571, by Abcam (1 mentions) Ab32503, by Abcam (1 mentions) Ap0063, by Bioworld Technology (1 mentions)

10 protocols using ab87540

Saponin-Mediated Autophagy Regulation

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SLSP was purchased from Qidan Co. Ltd. (purity > 95%, Wenshan, Yunnan, China). The main saponins of SLSP were ginsenoside Rb1 (4.86%), ginsenoside Rc (11.1%), notoginsenoside Fc (11.8%), ginsenoside Rb3 (17.4%), notoginsenoside FP2 (5.59%), notoginsenoside Fa (4.13%), ginsenoside Rd (3.04%), notoginsenoside IX (7.72%), notoginsenoside Fe (5.45%), and gypenoside XVII (3.67%).
Antibodies against Beclin-1(Cat. No. 3495, Rabbit mAb, 1:1000), LC3B (3868, Rabbit mAb, 1:1000), phospho–phosphoinositide 3-kinase (PI3K) p85 (4228, Rabbit mAb, 1:1000), phospho-Akt (9271, Rabbit mAb, 1:1000), PI3K p85 (4257, Rabbit mAb, 1:1000), Akt (9272, Rabbit mAb, 1:1000), Bcl-2 (2870, Rabbit mAb, 1:1000), Bax (2772, Rabbit mAb, 1:1000), and cleaved caspase-3 (9664, Rabbit mAb, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against p62 (ab109012, Rabbit mAb, 1:1000), p-mTOR (ab84400, Rabbit mAb, 1:1000), mTOR (ab87540, Rabbit mAb, 1:1000), and β-actin (ab8227, Rabbit mAb, 1:1000) were purchased from Abcam (Cambridge, MA, USA). Rapamycin and 3-methyladenine (3-MA) were provided by Selleckchem (Boston, MA, USA). Akt inhibitor VIII were bought from EMD Chemicals (An Affiliate of Merck KGaA, Darmstadt, Germany). All the other reagents were of analytical grade unless mentioned otherwise.

Cao Y., Yang Y., Wu H., Lu Y., Wu S., Liu L., Wang C., Huang F., Shi H., Zhang B., Wu X, & Wang Z. (2019). Stem-leaf saponins from Panax notoginseng counteract aberrant autophagy and apoptosis in hippocampal neurons of mice with cognitive impairment induced by sleep deprivation. Journal of Ginseng Research, 44(3), 442-452.

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Western Blotting for Cellular Signaling

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Western blotting was performed with the SDS-PAGE electrophoresis system. Adherent cells or adipose tissue extracts were prepared and transferred to PVDF membranes. The following primary antibodies were used: anti-GAPDH (ap0063, Bioworld), anti-Col15α1 (ab150463, Abcam), anti-Caspase-9 (ab32539, Abcam), anti-Cleaved Caspase-9 (bs7070, Bioworld), anti-Caspase-3 (Bs6428, Bioworld), anti-Cleaved Caspase-3 (bs7004, Bioworld), anti-Bcl2 (bs1511, Bioworld), anti-Bax (ab32503, Abcam), anti-AMPK (ab32047, Abcam), anti-pAMPK (ab133448, Abcam), anti-mTOR (ab87540, Abcam), anti-pmTORC1^Ser2448 (ab109268, Abcam), anti-Akt (ab8805, Abcam), anti-pAkt^Ser473 (ab18206, Abcam), anti-S6K1 (ab32529, Abcam), anti-pERK1/2 (ab201015, Abcam), anti-ERK1/2 (ab17942, Abcam), anti-pS6K1^Thr389 (ab2571, Abcam), anti-Collagen I (ab34710, Abcam), anti-Collagen VI (ab6588, Abcam), anti-Fibronectin (ab2413, Abcam), anti-MMP2 (ab92536, Abcam), anti-MMP9 (ab38898, Abcam), anti-TIMP1 (WL02342, Wanleibio), anti-TIMP2 (ab180630, Abcam), anti-FGFR1 (ab31324, Abcam), anti-pFGFR1^{Tyr653/Tyr654} (GTX133526, GeneTex), anti-TGFβ1 (WL03092, Wanleibio). Horseradish peroxidase anti-rabbit or anti-goat (Sigma–Aldrich) were used as secondary antibodies.

Xia T., Shen Z., Cai J., Pan M, & Sun C. (2020). ColXV Aggravates Adipocyte Apoptosis by Facilitating Abnormal Extracellular Matrix Remodeling in Mice. International Journal of Molecular Sciences, 21(3), 959.

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Western Blot Analysis of Metabolic Regulators

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Western blot was performed as previously described (22 (link)) by using the following antibodies: PGC-1α (P-19; sc-5815, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), nuclear respiratory factor-1 (NRF-1; ab55744, 1:500; Abcam, Cambridge, United Kingdom), transcription factor A, mitochondrial (TFAM; ab119684, 1:500; Abcam), AMPK (2532, 1:1000; Cell Signaling Technology, Danvers, MA, USA), phospho-AMPK-α for the detection of endogenous AMPKα only when phosphorylated at threonine 172 (2531, 1:1000: Cell Signaling Technology), serine–threonine liver kinase B1 (LKB1; ab15095, 1:100; Abcam), and mammalian target of rapamycin (mTOR; ab87540, 1:200; Abcam).

Imasawa T., Obre E., Bellance N., Lavie J., Imasawa T., Rigothier C., Delmas Y., Combe C., Lacombe D., Benard G., Claverol S., Bonneu M, & Rossignol R. (2016). High glucose repatterns human podocyte energy metabolism during differentiation and diabetic nephropathy. The FASEB Journal, 31(1), 294-307.

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Protein Expression Analysis of White Adipocytes

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Protein from white adipocytes or white adipose tissue was extracted using lysing buffer. Protein concentration was determined using BCA Protein Assay kit (Beyotime, Nanjing, China). The experimental procedure was as described previously [46 (link)]. Briefly, Protein samples (30 μg) were separated by SDS-PAGE and transferred to PVDF nitrocellulose membranes (Millipore, USA), and blocked with 5% Skim Milk Powder/Tween 20/TBST at room temperature for 2 h. Then, primary antibodies against Bcl-2 (bs1511), Cleaved Caspase-9 (bs7070), Cleaved Caspase-3 (bs7004), Gapdh (ap0063), β-actin (ap0060) (Bioworld, CA, USA), Hoxa5 (ab140636), Bax (ab32503), Cytochrome C (ab53056), Akt (ab8805), p-Akt^Ser473 (ab81283), mTOR (ab87540), p-mTORC1^Ser2448(ab137133), and S6K1 (ab9366), p-S6K1^Thr389 (ab2571) (Abcam, Cambridge, UK) were used to incubate the membranes at 4 °C overnight and the appropriate HRP-conjugated secondary antibodies (Boaoshen, China) were used for 2 h at room temperature. Akt phosphorylation-specific inhibitor MK2206 and mTORC1 inhibitor rapamycin were purchased from Selleck Chemical (USA). Proteins were visualized using chemiluminescent peroxidase substrate (Millipore, USA), and then the blots were quantified using ChemiDoc XRS system (Bio-Rad, USA).

Feng F., Ren Q., Wu S., Saeed M, & Sun C. (2017). Hoxa5 increases mitochondrial apoptosis by inhibiting Akt/mTORC1/S6K1 pathway in mice white adipocytes. Oncotarget, 8(56), 95332-95345.

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Immunohistochemical Analysis of Colon Tumor Xenografts

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Colon tumors from xenograft mice were fixed using formaldehyde (10%) at room temperature for 24 h and were then embedded in paraffin. The tissues were cut into 4-µm sections and mounted on glass slides. The sections were rinsed with PBS and placed in a solution containing primary antibodies directed against Ki67 (1:1,000; ab16667; Abcam), PCNA (1:1,000; ab18197; Abcam), Caspase3 (1:2,000; ab90437; Abcam), Caspase9 (1:1,000; ab32539; Abcam), Bcl-2 (1:1,000; ab692; Abcam), ERK (1:2,000; ab196883; Abcam), JNK (1:1,000; ab179461; Abcam), mTOR (1:1,000; ab87540; Abcam) and AKT (1:1,000; ab8978; Abcam) incubated overnight at 4°C. Subsequently, sections were incubated with the appropriate secondary antibodies, mouse primary IgG (1:1,500; ab6785; Abcam) and rabbit primary IgG (1:1,500; ab6721; Abcam), for 2 h at room temperature were added to specimens prior to visualization. The sections were then washed with PBS and observed by fluorescent video microscopy (BZ-9000; Keyence Corporation, Osaka, Japan) and a Ventana BenchMark automated staining system (Roche Applied Science, Penzberg, Germany) was used for observation of protein expression levels. A negative control was employed wherein mice were injected with 100 µl PBS.

You S., Li W, & Guan Y. (2018). Tunicamycin inhibits colon carcinoma growth and aggressiveness via modulation of the ERK-JNK-mediated AKT/mTOR signaling pathway. Molecular Medicine Reports, 17(3), 4203-4212.

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Isolation and Characterization of Rat CD8+ Tem Cells

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Peripheral blood mono-nuclear cells (PBMC) were isolated from blood samples and counted by flow cytometry. Cells were then labeled with the following fluorescence-conjugated monoclonal antibodies: anti-Rat CD45 PE (12-0451-81, eBioscience, San Diego, CA, USA), anti-Rat CD8a APC (17-0081-81, eBioscience), anti-Rat CD44 PE (25-0441-81, eBioscience), anti-Rat CD62L (104432, Biolegend). CD8⁺Tem cells were sorted by flow cytometry (EPICS-XL, Beckman-Coulter, USA), then stained for IFN-γ (11-7311-81, eBioscience), mTOR (ab87540, Abcam, Cambridge, MA), S6K (ab32529, Abcam), T-bet (ab91109, Abcam), and Eomes (53-4875-82, eBioscience) expression.

Wang H., Li J., Han Q., Yang F., Xiao Y., Xiao M., Xu Y., Su L., Cui N, & Liu D. (2017). IL-12 Influence mTOR to Modulate CD8+ T Cells Differentiation through T-bet and Eomesodermin in Response to Invasive Pulmonary Aspergillosis. International Journal of Medical Sciences, 14(10), 977-983.

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CD8+ Tem Cell Phenotyping by Flow Cytometry

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We used flow cytometry to isolate and count CD8⁺ Tem cells. Cells were labeled with the following fluorescently conjugated monoclonal antibodies: anti-mouse CD45-PE (12045181, eBioscience, San Diego, CA, USA), anti-mouse CD8a-APC (17008181, eBioscience), anti-mouse CD44-PE (25044181, eBioscience), and anti-mouse CD62L (104432, BioLegend, San Diego, CA, USA). After the CD8⁺ Tem cells were sorted by flow cytometry, they were stained with antibodies against mTOR (ab87540, Abcam, Cambridge, MA, USA), interferon γ (IFN-γ) (11731181, eBioscience), T-bet (ab91109, Abcam), S6K (ab32529, Abcam) and Eomes (53-4875-82, eBioscience).

Wang H., Xiao Y., Su L., Cui N, & Liu D. (2018). mTOR Modulates CD8+ T Cell Differentiation in Mice with Invasive Pulmonary Aspergillosis. Open Life Sciences, 13, 129-136.

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Isolation and Characterization of CD8+ Tem Cells

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Peripheral blood mononuclear cells were isolated from blood samples and counted by flow cytometry. Cells were then labeled with the following fluorescence-conjugated monoclonal antibodies: anti-rat CD45 PE (12-0451-81, eBioscience, San Diego, CA, USA), anti-rat CD8a APC (17-0081-81, eBioscience), anti-rat CD44 PE (25-0441-81, eBioscience), and anti-rat CD62L (104432, Biolegend). CD8⁺ Tem were sorted by flow cytometry (EPICS-XL, Beckman-Coulter, Indianapolis, IN, USA) and then stained for interferon (IFN)-γ (11-7311-81, eBioscience), mTOR (ab87540, Abcam, Cambridge, MA, USA), and S6K (ab32529, Abcam) expression.

Cui N., Wang H., Su L.X., Zhang J.H., Long Y, & Liu D.W. (2017). Role of Triggering Receptor Expressed on Myeloid Cell-1 Expression in Mammalian Target of Rapamycin Modulation of CD8+ T-cell Differentiation during the Immune Response to Invasive Pulmonary Aspergillosis. Chinese Medical Journal, 130(10), 1211-1217.

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Rapamycin Inhibits mTOR and p-mTOR in KYSE150 Cells

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KYSE150 cells were incubated with rapamycin (VETEC V900930, Sigma, USA) at 0, 10, 20, 50, or 100 nmol/L for 0, 2, 6, 12, or 24 hours to determine the inhibition of mTOR and p-mTOR by coimmunofluorescence. After the cells were fixed with paraformaldehyde, they were incubated with primary antibodies against mTOR (anti-mTOR antibody [53E11], mouse monoclonal, Abcam ab87540, USA) and p-mTOR (anti-mTOR [phospho-S2448], rabbit polyclonal, Abcam ab84400, USA) followed by treatment with secondary antibodies (donkey anti-mouse IgG [H+L] highly cross-adsorbed secondary antibody, Alexa Fluor 488; donkey anti-rabbit secondary antibody, Alexa Fluor 594; Invitrogen, USA). Fluorescence was observed on an Olympus IX 71 (Olympus, Japan) microscope.

Xiaoyu H., Yiru Y., Shuisheng S., Keyan C., Zixing Y., Shanglin C., Yuan W., Dongming C., Wangliang Z., Xudong B, & Jie M. (2018). The mTOR Pathway Regulates PKM2 to Affect Glycolysis in Esophageal Squamous Cell Carcinoma. Technology in Cancer Research & Treatment, 17, 1533033818780063.

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Western Blot Analysis of Apoptosis and Signaling Pathways

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Total proteins were separated from cultured cells and analyzed by Western blotting as performed as previously reported protocols [29] . The membranes were probed with speci c primary antibodies against Bcl2 (CST, #3498, 1:1000), Bax (CST, #2772, 1:1000), PI3K (CST, #4257, 1:1000), AKT (CST, #4691, 1:1000), p-mTOR (CST, #5536, 1:1000), mTOR (Abcam, ab87540, 1:1000), cleaved caspase-3 (Abcam, ab2302,1:1000), Ki67 (A nity Biosciences, AF0198, 1:1000), Nrf2 (A nity Biosciences, AF0639, 1:1000), HO-1 (A nity Biosciences, AF5393, 1:1000), p-PI3K (A nity Biosciences, AF3242, 1:1000), p-AKT (A nity Biosciences, AF0016, 1:1000), HIF-1α (A nity Biosciences, AF1009, 1:1000), LDHA (A nity Biosciences, DF6280, 1:1000), and β-actin (Proteintech, 66009-1-Ig, 1:2000) overnight at 4 °C. The addition of ECL substrate (Thermo Fisher Scienti c) was used for visualization. Lastly, the protein membranes were analyzed using Image-Pro Plus software (version 6.0) and normalized with β-actin protein levels.

Zhu X., Duan F., Wang Y., Zhang H., Wang X., Zhang Y., Tang H., Huang H., Chen B., & Sun L. (2021). Agrimoniin Sensitizes Pancreatic Cancer to Apoptosis through ROS-Mediated Energy Metabolism Dysfunction.

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