These assays were performed as described (Yang et al., 2007 (link)) using antibodies against GAPDH, β-actin, and caspase-3 (Cell Signaling Technology Inc., MA, USA); and Na+/K+-ATPase α1 (Abcam Inc. Cambridge, UK). Tylophorine and Reevesiosides A-I were prepared as described (Chang et al., 2013 (link), Yang et al., 2010 (link)). DMSO (D1435, ≧ 99.5%), digoxin (D6003, ≧ 95%, HPLC), digitoxin (D5878, ≧ 92%, HPLC), digitoxigenin (D9404, 99%, TLC), ouabain (O3125, ≧ 95%, HPLC), dihydroouabain (D0670, ≧ 98.5%, TLC), oleandrin (O9640, ≧ 98%, HPLC), N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (Db cAMP) (D0627, ≧ 96%, HPLC), and aldosterone (A9477, ≧ 95%, HPLC) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Digitoxigenin
Manufactured by Merck Group
Sourced in United States
Digitoxigenin is a cardiac glycoside compound used as a reference standard in laboratory analysis and research. It is a naturally occurring substance found in the foxglove plant (Digitalis purpurea). Digitoxigenin plays a role in the study and identification of cardiac glycosides, which are important in the development and evaluation of certain pharmaceutical products.
Automatically generated - may contain errors
Lab products found in correlation
Digoxin, by Merck Group (5 mentions)
Ouabain, by Merck Group (4 mentions)
Digitoxin, by Merck Group (2 mentions)
Oleandrin, by Merck Group (2 mentions)
Proscillaridin, by Merck Group (2 mentions)
Aldosterone, by Merck Group (1 mentions)
Dihydroouabain, by Merck Group (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
N6 2 o dibutyryladenosine 3 5 cyclic monophosphate sodium salt dbcamp, by Merck Group (1 mentions)
Na k atpase α1, by Abcam (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Diatomaceous earth, by Merck Group (1 mentions)
Acetonitrile acn, by Merck Group (1 mentions)
Milli q plus system, by Merck Group (1 mentions)
Odoroside a, by Merck Group (1 mentions)
Withanolide a, by Merck Group (1 mentions)
Bufalin, by Merck Group (1 mentions)
Crystal violet, by Merck Group (1 mentions)
7 protocols using digitoxigenin
1
Comprehensive Protein Assays and Compound Characterization
2
Quantitative Analysis of Cardiac Glycosides
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κ-Strophanthidin (STR, > 90% purity), digitoxigenin (DTG, > 97% purity), odoroside A (ODA, > 97% purity), bufalin (BUF, > 98% purity), withanolide A (WTH, > 95% purity), proscillaridin (PRO, > 80% purity) and digoxin (DGX, > 95% purity) were purchased from Sigma-Aldrich (Hamburg, Germany). Convallatoxin (CTX) was purchased from MP Biomedicals (Irvine, CA, USA). Individual standard solutions were prepared in MeOH at concentration of 500 mg L-1 for STR, DGX, WTH and 100 mg L-1 for DTG, ODR, PRO, BUF, CTX and stored at −20 °C. A working standard mixture (ST) was prepared by 1000 times dilution in 50% (v/v) aqueous methanol prior to analysis.
LC-MS grade formic acid (FA), methanol (MeOH) and acetonitrile (ACN) were purchased from Sigma-Aldrich (Søborg, Denmark). Millipore water was obtained from a Millipore Milli Q-Plus system (Bedford, MA, USA). Glass beads (0.25 – 0.5 mm) were purchased from Carl Roth (Roth, Germany). C18 resin (Sepra E-C18, 50 μm) was purchased from Phenomenex (Værløse, Denmark). Diatomaceous earth and Ottawa sand (20 – 30 mesh) were purchased from Sigma-Aldrich (St. Louis, MI, USA) and Restek (Bellefonte, PA, USA); both were burnt overnight at 450 °C before use.
LC-MS grade formic acid (FA), methanol (MeOH) and acetonitrile (ACN) were purchased from Sigma-Aldrich (Søborg, Denmark). Millipore water was obtained from a Millipore Milli Q-Plus system (Bedford, MA, USA). Glass beads (0.25 – 0.5 mm) were purchased from Carl Roth (Roth, Germany). C18 resin (Sepra E-C18, 50 μm) was purchased from Phenomenex (Værløse, Denmark). Diatomaceous earth and Ottawa sand (20 – 30 mesh) were purchased from Sigma-Aldrich (St. Louis, MI, USA) and Restek (Bellefonte, PA, USA); both were burnt overnight at 450 °C before use.
3
Antiviral Compound Characterization Protocol
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DMSO (≧99.5%), digoxin (D6003, ≧95%, HPLC), digitoxin (D5878, ≧92%, HPLC), digitoxigenin (D9404, 99%, TLC), ouabain (O3125, ≧95%, HPLC), oleandrin (O9640, ≧98%, HPLC), crystal violet (C0775, dye content ≥90%), and methylcellulose (#M0387) were purchased from Sigma-Aldrich (St. Louis, MO, United States); bufalin (15725, ≧ 98%, HPLC) from Cayman Chemical (Ann Arbor, MI, United States); digoxin-BSA (80-ID10, HPLC) from Fitzgerald Industries (Acton, MA, United States); rostafuroxin (T2621, ≧99%, HPLC) from Target Molecule Corp. (Boston, MA, United States); istaroxime hydrochloride (HY-15718A, ≧99%, HPLC) from MedChem Express (Monmouth Junction, NJ, United States); and Remdesivir (GS-5734) (S8932, 99.3%, HPLC) and GS-441524 (S6814, 99.3%, HPLC) were from Selleckchem (Houston, TX, United States). The antibody against nucleocapsid proteins of HCoV-OC43 (Mab9013) was purchased from Merck Millipore (Burlington, MA, United States), and fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin (#55499) from MP Biomedicals (Irvine, CA, United States). Anti-SARS-CoV-2 N protein antibodies were provided by Dr. An-Suei Yang of the Genomics Research Center, Academia Sinica. Goat anti-human IgG-Alexa Fluor 488 (A11013) and DAPI (D1306) were purchased from Invitrogen. 10% formaldehyde solution was purchased from Marcon™ Chemicals (#H121-08).
4
Autophagy Regulation by P2RX7 Signaling
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The following antibodies were used: anti-P2RX7 (Synaptic Systems, 177003) 1:1000, anti-MAPK1-MAPK3 (Cell Signaling Technology, 9102) 1:2000, anti-phospho-MAPK1-MAPK3 (Cell Signaling Technology, 9106) 1:1000, anti-ACTB (Sigma, A2066) 1:1000, anti-LC3B (Sigma, L7543) 1:1000, anti-GAPDH (Sigma, G9545) 1:1000, anti-COX4/COXIV (Abcam, ab14744) 1:000, anti-BECN1 (Santa Cruz Biotechnology, 48381) 1:200. Other chemicals used were as follows: P2RX7 antagonists A438079 and A804598 (Tocris Bioscience, 2972 and 4473, respectively) and Brilliant Blue G (BBG, Ascent Scientific, ASC-389), HSPA2 and HSP90 inhibitors, VER155008, geldanamycin and 17-DMAG (Tocris Biosciences, 3803, 1368 and 2610, respectively), Proteostat aggresome detection kit (Enzo Life Sciences, ENZ-51035-K100), HSPA2 inhibitor KNK437 (Merck Millipore, 373260), the cell permeable, fluorogenic CASP3-CASP7 substrate DEVD-Nucview488 (Cambridge Biosciences, BT10403) and the mitochondrial membrane potential-sensitive probe tetramethylrhodamine ethyl ester (TMRE; Sigma, 87917), digoxin and digitoxigenin (Sigma, D6770 and D9404, respectively).
5
Cane Toad Compound Isolation and Identification
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Marinobufagin (1 ), marinobufotoxin (6 ) and suberoyl-l -arginine (13 ) were obtained from our in-house pure compound library, and their purities were confirmed by LCMS, HRMS and NMR (see Supporting Information for 1H NMR spectra of the pure compounds). Plant cardenolides: digitoxigenin (14 ), ouabain (15 ) and digoxin (16 ) (Fig. 2 ) were purchased from Sigma Aldrich and were used in the attractant assay without further purification.![]()
Compounds identified in different stages of cane toad (Rhinella marina) (
6
Colorimetric Assays for Metabolic Activity
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Proscillaridin, ouabain, digitoxigenin, digoxin, withaferin-A, and lanatoside-C were purchased from Sigma-Aldrich (St. Louis, MO). ATP colorimetric assay, glucose colorimetric assay, and lactate colorimetric assay kits were purchased from BioVision (Milpitas, CA). CellROX Deep Red Reagent was purchased from Invitrogen.
7
Digitoxigenin Formulation for Bioavailability
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Digitoxigenin and olive oil (highly refined, low acidity) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alginic acid sodium salt (low viscosity) was from MP Biomedicals (Solon, OH, USA). Tween 85 (Acros Organics, Morris, NJ, USA) and Span 85 (Sigma-Aldrich, St. Louis, MO, USA) were used in the preparation of Digitoxigenin formulations. Other reagents used were isooctane (Acros Organics, Morris, NJ, USA) and calcium chloride dehydrate (Fisher Scientific, Springfield, NJ, USA). Phosphate buffered saline (PBS) of pH 7.4 was prepared by dissolving PBS tablets (MP Biomedicals, Solon, OH, USA) in deionized water, and sodium azide (Acros Organics, Morris, NJ, USA) was added at a concentration of 0.02% (w/v) as a bacteriostat. High performance liquid chromatography (HPLC) grade methanol, water, and formic acid were purchased from Fisher Scientific (Pittsburgh, PA, USA).
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