Eht 1864

Manufactured by R&D Systems

The EHT 1864 is a laboratory equipment designed for high-voltage electrophoresis. It is capable of generating a stable and adjustable high-voltage electric field, which is a key requirement for various electrophoresis-based applications in research and analytical laboratories.

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5 protocols using eht 1864

Quantitative Analysis of Cellular Signaling

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Antibodies directed against total (Catalog# 2524) and phosphorylated (Serine-15) p53 (Catalog# 9284) were from Cell Signaling Technology (Danvers, MA). Antisera against total (Catalog# ab78) and phosphorylated (Serine-1981) ATM kinase (Catalog# ab36810) were obtained from Abcam (Cambridge, MA). Published evidence from several laboratories have confirmed the specificity of antibodies utilized in this study [30 (link), 33 (link)]. IRDye® 800CW anti-rabbit and anti-mouse were obtained from LICOR (Lincoln, NE). KU-55933, Simvastatin and Cell Death Detection ELISA® kit were from Sigma (St. Louis, MO). EHT1864 and SB203580 were from R&D Systems (Minneapolis, MN). GGTI-2147 was purchased from VDM Biochemicals (Bedford, OH). NE-PER® Nuclear and Cytoplasmic Extraction Reagents were from Thermo Scientific (Waltham, MA).

Sidarala V, & Kowluru A. (2017). Exposure to chronic hyperglycemic conditions results in Ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated activation of p53 and ATM kinase in pancreatic β-cells. Apoptosis : an international journal on programmed cell death, 22(5), 597-607.

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Antibody-based Immunoblotting Assay

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Rabbit polyclonal antibody for phospho-p38MAPK (Thr 180/Tyr 182) and total-p38MAPK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cleaved caspase-3 and lamin B antibodies were purchased from Cell Signaling (Danvers, MA). Actin antibody was from Sigma–Aldrich (St. Louis, MO). Anti-rabbit IgG-horseradish peroxidase conjugate and enhanced chemiluminescence (ECL) kits were from Amersham Biosciences (Piscataway, NJ). IRDye® 800CW anti-rabbit was obtained from LICOR (Lincoln, NE). NSC23766 and GGTI-2147 were purchased from Calbiochem (San Diego, CA). Ehop-016 was synthesized as we described in [20 (link)]. EHT1864 was from R&D systems (Minneapolis, MN). Scrambled gp91-ds-tat (inactive) and active gp91-ds-tat were from Anaspec, Inc. (Fremont, CA). 2-Bromo-palmitate (2-BP), 2′,7′-dichlorofluorescein diacetate (DCFDA), N,-N′-dimethyl-9,−9′-bisacridiniumdinitrate (lucigenin), were from Sigma–Aldrich (St. Louis, MO). The rat insulin ELISA kit was purchased from American Laboratory Products Co (Wind-ham, NH). All other reagents used in the studies were obtained from Sigma–Aldrich (St. Louis, MO).

Sidarala V., Veluthakal R., Syeda K., Vlaar C., Newsholme P, & Kowluru A. (2015). Phagocyte-like NADPH oxidase (Nox2) promotes activation of p38MAPK in pancreatic β-cells under glucotoxic conditions: Evidence for a requisite role of Ras-related C3 botulinum toxin substrate 1 (Rac1). Biochemical pharmacology, 95(4), 301-310.

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Integrin and Cell Signaling Inhibition

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The antibodies, anti-human IgG (Fc) (BioConcept AG, Switzerland), AIIB2 (integrin β₁-subunit blocking antibody), P5H9 (α_vβ₅-integrin blocking antibody) and P1B5 (integrin α₃-subunit blocking antibody; all 10 μg/mL supernatants; DSHB, Iowa) were incubated with cells on ice for 30 minutes prior to SCFS. To inhibit cell signaling, wortmannin (100 nM; Sigma-Aldrich)47 (link), LY294002 (50 μM; Cell Signaling Technology)48 (link), NSC23766 (50 μM; Merck Millipore)49 (link), EHT 1864 (100 μM; R&D Systems)50 (link), GGTI 2147 or GGTI286 (10 μM; Merck Millipore)51 (link), Akt inhibitor IV (1 μM; Merck Millipore)52 (link), Akt inhibitor VIII (20 μM; Merck Millipore)53 (link), SecinH3 (20 μM; Merck Millipore)54 (link), blebbistatin (10 μM; Merck Millipore)55 (link) or Y-27632 (10 μM; Merck Millipore)57 (link) were added to the measurement media at the concentrations given. Cells were then incubated for 30 minutes at 37°C prior to SCFS. All inhibitors were dissolved in DMSO and stored at −20°C.

Yu M., Wang J., Muller D.J, & Helenius J. (2015). In PC3 prostate cancer cells ephrin receptors crosstalk to β1-integrins to strengthen adhesion to collagen type I. Scientific Reports, 5, 8206.

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Phospho-p38MAPK and CD36 Immunoblotting

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Rabbit polyclonal antibody for phospho-p38MAPK [Thr 180/Tyr 182] and total-p38MAPK and mouse monoclonal antibody for CD36 were from Santa Cruz Biotechnology [Santa Cruz, CA]. Phospho-p53, total-p53 and cleaved caspase-3 antibodies were from Cell Signaling [Danvers, MA]. IRDye^® 800CW anti-rabbit and anti-mouse secondary antibodies were from LICOR [Lincoln, NE]. Metformin hydrochloride was purchased from Sigma-Aldrich. Rac1 antiserum was from BD Transduction Laboratories [San Jose, CA]. Antisera against cleaved caspase-3, lamin B, Phospho and total p53 and Myc tag were from Cell Signaling [Danvers, MA]. Mouse monoclonal β-actin antibody was obtained from Sigma-Aldrich [St. Louis, MO]. GGTI-2147 was from VDM biochemicals [Bedford Heights, OH]. EHT 1864 was from R&D Systems [Minneapolis, MN]. NE-PER^® Nuclear and cytoplasmic extraction kit was from Thermo Scientific [Waltham, CA]. Lipofectamine 3000 was from Invitrogen [Carlsbad, CA]. All other reagents used in the studies were obtained from Sigma-Aldrich [St. Louis, MO].

Baidwan S., Chekuri A., Hynds D.L, & Kowluru A. (2017). Glucotoxicity promotes aberrant activation and mislocalization of Ras-related C3 botulinum toxin substrate 1 [Rac1] and metabolic dysfunction in pancreatic islet β-cells: Reversal of such metabolic defects by metformin. Apoptosis : an international journal on programmed cell death, 22(11), 1380-1393.

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Signaling Pathway Analysis Protocol

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EHT 1864 and PD0325901 were obtained from R&D systems (Minneapolis, MN). Simvastatin was from Sigma (St. Louis, MO). The rat insulin ELISA kit was purchased from American Laboratory Products Co (Windham, NH). Rac1 activation G-LISA kit was from Cytoskeleton Inc. (Denver, CO). Antibodies against phospho-p44/42 ERK1/2 (Thr202/Tyr204), total p44/42 ERK1/2, phospho-Akt (Ser473), total Akt, phospho-p53 (Ser15) and E-Cadherin were obtained from Cell Signaling Technology (Danvers, MA). Antisera directed against GAPDH and total p53 were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibody directed against Rac1 was purchased from BD Bioscience (San Jose, CA).

Sidarala V., Veluthakal R., Syeda K, & Kowluru A. (2015). EHT 1864, a small molecule inhibitor of Ras-related C3 botulinum toxin substrate 1 (Rac1), attenuates glucose-stimulated insulin secretion in pancreatic β-cells. Cellular signalling, 27(6), 1159-1167.

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