The screened compounds were purchased from the SPECS database and PKU-CNCL. The purities of all purchased compounds were more than 90%. For the chemical synthesis, reagents and solvents were obtained from J&K Chemical (Beijing, China) without further purification. T16Ainh-A01 was purchased from TOCRIS (USA). 1H-NMR and 13C-NMR spectra were recorded on Bruker (400 MHz) instruments, using DMSO-d6 or CDCl3 as solvents. High-resolution mass spectra (HRMS) were recorded on the Bruker Apex IV FTMS mass spectrometer using ESI (electrospray ionization). The microwave-assisted reaction was performed on the CEM Discover SP microwave reactor. The purity of the final compound was determined by NMR and HPLC to be >95% (UV detection (λ = 254 nm), compound structures in smiles format and more detail can be found in Supporting Information ).
T16ainh a01
Manufactured by Bio-Techne
Sourced in United Kingdom, United States
The T16Ainh-A01 is a lab equipment product from Bio-Techne. It is designed for use in laboratory settings. The core function of this product is to [description not available].
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Lab products found in correlation
Mecamylamine, by Merck Group (1 mentions)
Dantrolene, by Bio-Techne (1 mentions)
Papaine, by Merck Group (1 mentions)
Chelerythrine chloride, by Bio-Techne (1 mentions)
Epibatidine, by Bio-Techne (1 mentions)
Leupeptine, by Merck Group (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
α bungarotoxin, by Bio-Techne (1 mentions)
Bovine serum albumin (bsa), by Carl Roth (1 mentions)
Tween 20, by Avantor (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Caccinh a01, by Bio-Techne (1 mentions)
Calcium d gluconate, by Merck Group (1 mentions)
Tyrphostin ag490 ag 490, by Merck Group (1 mentions)
α conotoxin mii, by Bio-Techne (1 mentions)
Dihydro β erythroidine dhβe, by Bio-Techne (1 mentions)
Chromanol 293b, by Bio-Techne (1 mentions)
Thapsigargin, by Cayman Chemical (1 mentions)
Wp1066, by Cayman Chemical (1 mentions)
Mgcl2, by Merck Group (1 mentions)
10 protocols using t16ainh a01
1
Synthesis and Characterization of T16Ainh-A01 Compound
2
Pharmacological Modulation of Nicotinic Receptors
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d ‐glucose, calcium d ‐gluconate, DMSO, ACh, ATP, mecamylamine , NaHCO3, papaine, l ‐cysteine, leupeptine, pyruvic acid, NaOH and tyrphostin AG490 (AG 490) were acquired from Sigma‐Aldrich (Taufkirchen, Germany). A‐85380 , α‐bungarotoxin , α‐conotoxin PnIA , α‐conotoxin MII , chelerythrine chloride, dihydro‐β‐erythroidine (DHβE ), epibatidine , T16Ainh‐A01, Ani 9, CaCCinh‐A01, Chromanol293B, ACV 1 (conotoxin Vc1.1), dantrolene , JTV519 and H‐89 were obtained from Tocris Bioscience (Abingdon, UK). The α‐conotoxin ImI was ordered from Alomone Labs (Jerusalem, Israel). KH2PO4, KCl and MgCl2 were purchased from MERCK (Darmstadt, Germany). NaCl was ordered from Grüssing GmbH (Westoverledingen, Germany). HEPES and BSA were from Carl Roth (Karlsruhe, Germany). Thapsigargin and WP1066 were acquired from Cayman Chemicals (Ann Arbor, MI, USA) and nicotine from Glentham Life Sciences (Corsham, UK). EDTA and Tween 20 were obtained from VWR (Darmstadt, Germany). Sodium pyruvate was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA) and DNase1 from Invitrogen (Thermo Fisher Scientific).
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3
Calcium Imaging with Pharmacological Inhibitors
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Muscles used for Ca2+ imaging were perfused constantly with KRB solution containing (mmol/L): NaCl, 120.35; KCl, 5.9; NaHCO3, 15.5; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.5; and glucose, 11.5. KRB solution was bubbled with a mixture of 97% O2 – 3% CO2 and warmed to 37 ± 0.2°C. Drugs used to inhibit CaCC were 5-nitro-2-(3-phenylpropylamino)-benzoic acid [NPPB; purchased from Sigma; (Yang et al., 2008 (link))] and 2-[(5-Ethyl-1,6-dihydro-4-methyl-6-oxo-2-pyrimidinyl)thio]-N-[4-(4-methoxyphenyl)-2 thiazolyl]acetamide [T16Ainh-A01; purchased from Tocris Bioscience (Ellisville, MO, United States); (Namkung et al., 2011 (link))]. Drugs used to inhibit NCX were 2-[2-[4-(4-Nitrobenzyloxy) phenyl]ethyl] isothiourea mesylate [KB-R7943; purchased from Tocris Bioscience; (Watano et al., 1996 (link))] and 2-[[4-[(4-Nitrophenyl)methoxy]phenyl]methyl]-4-thiazolidinecarboxylic acid ethyl ester [SN-6; purchased from Tocris Bioscience; (Niu et al., 2007 (link))]. NPPB (100 mM), T16Ainh-A01 (50 mM), KB-R7943 (10 mM) and SN-6 (10 mM) were dissolved in dimethyl sulphoxide (DMSO). Final dilutions were made by adding stock solutions to extracellular solutions used for specific experiments. Final concentrations of DMSO were less than 0.1%.
4
Inhibition of Anoctamin-1 and Calcium-Activated Chloride Channels
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5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), anthracene-9-carboxylic acid (9-AC) and niflumic acid (NFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The specific anoctamin-1 inhibitor (T16Ainh-A01) was purchased from Tocris Bioscience (Avon, Bristol, England). The specific calcium-activated chloride channel inhibitor (CaCCinh-A01) was purchased from Merck Millipore (Billerica, MA, USA). All drugs were dissolved in 30% dimethyl sulfoxide (DMSO) in all doses tested.
5
Screening of Small Molecule Modulators
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T16Ainh-A01 (Tocris), digallic acid (Toronto Research Chemicals), pentagalloyl glucose (PGG) (Sigma-Aldrich), tannic acid (Bosche Scientific), CaCCinh-NV2 and -NV7, brefeldin A (BFA), chloroquine, MG132 (Sigma-Aldrich), cycloheximide (Calbiochem), CaCCinh-A01 (Specs), CaCCinh-NV4 (ChemDiv), and all other compounds obtained from our internal archive were dissolved in DMSO to a final concentration of 10 mm . If not noted otherwise, cells were treated with 10 μm compound.
6
Compound Screening for Ion Channel Modulators
7
IL-13 and TMEM16A Inhibitor Effects
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Medium containing IL-13 (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) was added to the basolateral side of the inserts from beginning of ALI culture for 14 days as described before.27 (link),28 (link) Likewise, medium containing T16Ainh-A01 (10 µM; Tocris, Bristol, UK), a TMEM16A inhibitor, was added in the same manner. Control wells were cultured with 0.5 mL of ALI medium alone.
8
Whole-Cell Patch Clamp Experiments
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For the whole-cell patch clamp experiment, low Ca2+-buffered KCl pipette solution contained (in mM): 140 KCl, 10 HEPES, 5 NaCl, 3 Mg-ATP, 0.1 EGTA, 1 MgCl2 and was adjusted to pH 7.2 with KOH. Ca2+-clamped KCl pipette solution contained (in mM) 140 KCl, 1 MgCl2, 3 Mg-ATP, 10 HEPES, 5 EGTA, 5 NaCl, and 300 nM free Ca2+ calculated using the free WebMax software (www.stanford.edu/~cpatton/webmaxcS ), and was adjusted to pH 7.25 with KOH. Ca2+-clamped CsCl pipette solution contained (in mM): 140 CsCl, 1 MgCl2, 3 Mg-ATP, 10 HEPES, 5 EGTA, 5 NaCl, and 600 nM free Ca2+calculated using the free WebMax software and was adjusted to pH 7.25 with CsOH. Normal Tyrode's (NT) bath solution contained (in mM): 145 NaCl, 3.6 KCl, 1 MgCl2, 1.3 CaCl2, 10 HEPES, 5 glucose, 10 sucrose and was adjusted to pH 7.4 with NaOH. NMDG-Cl bath solution contained: 150 NMDG-Cl, 10 HEPES, 10 glucose, 1 MgCl2, 1.3 CaCl2 and was adjusted to pH 7.4 with NMDG-OH. Ionomycin, 1-ethyl-2-benzimidazolinone (1-EBIO) and 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) were purchased from Sigma-Aldrich. T16Ainh-A01, a specific inhibitor of Ano-1, was purchased from TOCRIS Biosciences. The chemicals were initially dissolved in dimethyl sulfoxide (DMSO), and were diluted to the bath solution for experiments. The amount of DMSO in the bath solution was kept below 0.1%.
9
Adult Mouse Salivary Gland Analysis
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Male C57BL/6J adult mice (8 weeks of age) were purchased from CLEA Japan (Tokyo, Japan). Mice were maintained under a 12-h light/dark cycle and fed ad libitum. All experiments were approved by the Animal Committee of Kyushu Dental University (No. 16–011). Collagenase L was purchased from Nitta Gelatin (Osaka, Japan); Fura 2-AM was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan); 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) was purchased from Life Technologies Corporation (Eugene, OR, USA); T16Ainh-A01 was obtained from Tocris Bioscience (Bristol, UK); the NKCC1 antibody (sc-21545) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA); the TMEM16A antibody (ab53213) was purchased from Abcam (Cambridge, UK); the AQP5 antibody (sc-9890) was purchased from EMD Millipore (Billerica, MA, USA); and the secondary antibody (Histofine Simple Stain Mouse MAX peroxidase) was purchased from Nichirei (Tokyo, Japan). All other reagents were purchased from Sigma-Aldrich Japan (Tokyo, Japan).
10
Investigating Heat Hyperalgesia in Tongue
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We assessed the involvement of TRPM3 and anoctamin 1 (Ano1) in heat hyperalgesia of the tongue induced by CFA injection. The TRPM3 inhibitor (1 µL, 1 mM; Liquiritigenin, Sigma-Aldrich, St. Louis, MO) and the Ano1 inhibitor (1 µL, 1 mM; T16Ainh-A01, Tocris Bioscience, Bristol, UK) were dissolved in 50% dimethylsulfoxide in physiological saline. On day 3 after CFA injection into the tongue, HHWTs were measured before and 30, 60, 90 and 120 min after tongue administration of Liquiritigenin or T16Ainh-A01 as described earlier. Before CFA injection into the tongue, prevalue of HHWTs was measured in advance.
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