Ana 12

Manufactured by Bio-Techne

Sourced in United States, United Kingdom

ANA-12 is a small molecule tyrosine phosphatase inhibitor. It is used as a research tool to study the role of the tyrosine phosphatase SHP2 in cellular processes.

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20 protocols using ana 12

ANA12 and SGN Cell Signaling

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SGN samples were dissected and plated as described above. After the tissues had adhered to the plate for 5 h at 37 °C, 5% CO₂, SGN samples receiving ANA12 had media changed to 50 «L of culture medium containing ANA12 (Tocris Bioscience) at a final concentration of 10 nM. Control samples were incubated overnight without ANA12. ANA12 was stored as stock solution in DMSO at 10 μM at −20 °C. All tissues in both plates were incubated overnight at 37 °C, 5% CO2.
After 24 h, all cultures received 400 nM of DHF, Ris, Ris-DHF, or DMSO alone (control) as described above. SGNs in the ANA12 assay plate additionally received 10 nM ANA12 in culture medium. Cultures was kept at 37 °C, and media was changed every 24 h. After 48 h, the cells were fixed, permeabilized, stained, and evaluated with confocal microscopy as described above.

Kempfle J.S., Nguyen K., Hamadani C., Koen N., Edge A.S., Kashemirov B.A., Jung D.H, & McKenna C.E. (2018). Bisphosphonate-Linked TrkB Agonist: Cochlea-Targeted Delivery of a Neurotrophic Agent as a Strategy for the Treatment of Hearing Loss. Bioconjugate chemistry, 29(4), 1240-1250.

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Pharmacologically Induced Hypothermia in Mice

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C57Bl6/J wild-type mice, tg37^+/− (Mallucci et al, 2002 (link)) and B6;129S2-Ntrk2^tm1Bbd/J (Ntrk2^+/−) mice (https://www.jax.org/strain/002544) weighing ≥20 g were cooled using 5′-AMP as described (Peretti et al, 2015 (link)). Briefly, mice were intraperitoneally injected with freshly prepared 5′-AMP (0.7 mg per g; Sigma-Aldrich) or saline control. Mice were maintained at room temperature until core body temperature decreased to 25°C (∼60 min). Subsequently, mice were transferred to a refrigerator (5–10°C) and core body temperature lowered to 16–18°C for 45 min. For rewarming, mice were allowed to recover to normal body temperature at room temperature conditions. Cooled samples were collected at the end of the 16–18°C period and rewarmed samples as stated elsewhere in the text. ANA-12 (N-[2-[[(Hexahydro-2-oxo-1H-azepin-3- yl)amino]carbonyl]phenyl]benzo[b]thiophene-2-carboxamide) (Tocris) compound was intraperitoneally injected (0.5 mg/kg in 0.5% DMSO in saline solution) in wild-type and prion-infected mice, twice a day for 2 d before cooling and 2 h before the initiation of the cooling protocol. 7,8-Dihydroxyflavone (7,8-DHF) (Tocris Bioscience) was injected intraperitoneally to C57Bl6/J wild-type mice at 5 mg/kg in 17% DMSO in PBS buffer (Devi & Ohno, 2012 (link)).

Peretti D., Smith H.L., Verity N., Humoud I., de Weerd L., Swinden D.P., Hayes J, & Mallucci G.R. (2021). TrkB signaling regulates the cold-shock protein RBM3-mediated neuroprotection. Life Science Alliance, 4(4), e202000884.

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Neuroprotective Mechanisms of Sulfur Compounds

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Male Wistar albino rats (200-250 g) were used for this study, and these were maintained at standard laboratory conditions (40% humidity, a temperature of 24°C-26°C, and 12 hr of light and dark). The experimental protocol was approved by the Institutional Animal Ethics Committee of Tianjin First People’s Hospital (Ethical committee) with ethical approval number: 2019528A65F025. The experiments were performed as per the ethical guidelines. ANA-12, BDNF receptor blocker (Tocris Bioscience, Bristol, UK), diallyl disulfide (Sigma-Aldrich, St. Louis, Missouri) and diallyl trisulfide (Sigma-Aldrich) were used as pharmacological agents. The enzyme-linked immunosorbent assay (ELISA) kits for the quantification of BDNF and Nrf2 were procured from RayBiotech, Peachtree Corners, Georgia.

Wang G., Yang Y., Wang C., Huang J., Wang X., Liu Y, & Wang H. (2020). Exploring the role and mechanisms of diallyl trisulfide and diallyl disulfide in chronic constriction-induced neuropathic pain in rats. The Korean Journal of Pain, 33(3), 216-225.

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Experimental Stimulation of Neuronal Receptors

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Slices were incubated with 10 nM E₂ (EMD Millipore) or 100 nM G1 (Tocris Bioscience) at 37°C for the indicated periods of time. Alternatively, slices were incubated with 100 nM PPT (Tocris Bioscience) or 10 nM DPN (Tocris Bioscience) for 1 h. Treatment with DHPG (100 µM; Tocris Bioscience) was generally performed 1 h after G1 application and lasted 15 min, unless stated otherwise. In some experiments, slices were pretreated for 30 min with different inhibitors or antagonists, including ANA-12 (50 µM; Tocris Bioscience), cycloheximide (25 µM; Tocris Bioscience), MG132 (25 µM; EMD Millipore), rapamycin (1 µM; Cell Signaling Technology), and G36 (1 µM; Azano Biotech). TrkB-Fc (2 µg/ml; R&D Systems) or IgG-Fc (2 µg/ml; Bethyl Laboratories, Inc.) were preincubated for 1 h before chemical treatment.

Briz V., Liu Y., Zhu G., Bi X, & Baudry M. (2015). A novel form of synaptic plasticity in field CA3 of hippocampus requires GPER1 activation and BDNF release. The Journal of Cell Biology, 210(7), 1225-1237.

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Purification and Characterization of Chemicals

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Chemicals and reagents used for the experiments were obtained from Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Sigma Aldrich; and Merk KGaA, Darmstadt, Germany, unless stated otherwise. Thymol (purity > 99%) was purchased from Tokyo Chemical Industry Company Limited (TCI), Tokyo, Japan (Pubchem substance ID: 87572469). Stigmasterol (purity ~95%), nocodazole (purity ~99%), quercetin (purity > 95%) (Pubchem substance ID: 329823865), and paclitaxel (purity > 95%) (Pubchem substance ID: 24277878) were purchased from Sigma-Aldrich, Saint Louis, MO, USA. ANA-12 (Pubchem substance ID: 2799722) was purchased from from Tocris Biosciences, Fischer Scientific, USA.

Timalsina B., Haque M.N., Dash R., Choi H.J., Ghimire N, & Moon I.S. (2023). Neuronal Differentiation and Outgrowth Effect of Thymol in Trachyspermum ammi Seed Extract via BDNF/TrkB Signaling Pathway in Prenatal Maternal Supplementation and Primary Hippocampal Culture. International Journal of Molecular Sciences, 24(10), 8565.

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Pharmacological Regulation of Synaptic Signaling

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For activating mGluR5, CHPG (Cat. No. 1049 – Tocris Bio-Techne Corporation) a mGluR5 selective agonist was used at a dose of 1mM, whereas for inhibiting NMDAR function, (+)-MK 801 maleate (Cat. No. 0924 Tocris Bio-Techne Corporation) a selective non-competitive antagonist was added at a final concentration of 3uM. Lastly, ANA-12 (Cat. No. 4781 Tocris – BioTechne) that is a TrkB receptor antagonist was added at 1uM.
Pharmacological and psychotropic treatment experiments.

Mellios N., Papageorgiou G., Gorgievski V., Maxson G., Hernandez M., Otero M., Varangis M., Dell’Orco M., Perrone-Bizzozero N, & Tzavara E. (2024). Regulation of neuronal circHomer1 biogenesis by PKA/CREB/ERK-mediated pathways and effects of glutamate and dopamine receptor blockade. Research Square.

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TrkB Signaling Blockade and Electroacupuncture Modulate Cognitive Function

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Mice were randomly distributed into EA, ANA-12, and ANA-12+EA treatment groups (n = 7 per group). For blockade of TrkB signaling, the specific TrkB antagonist ANA-12 (0.5 mg/kg/day, 7 days, i.p.; Tocris Bioscience, Bristol, UK)43 (link) was administered 1 h prior to each EA session. EA was applied once daily for 7 consecutive days and MWM tests were performed 5 days after treatment initiation and 3 days after treatment completion. Experimental results were recorded using SMART, version 2.5.18 (Panlab S.L.U.). Mice were sacrificed and tissues were analyzed for MBP expression and BrdU incorporation after the final MWM test.

Ahn S.M., Kim Y.R., Kim H.N., Shin Y.I., Shin H.K, & Choi B.T. (2016). Electroacupuncture ameliorates memory impairments by enhancing oligodendrocyte regeneration in a mouse model of prolonged cerebral hypoperfusion. Scientific Reports, 6, 28646.

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Intrahippocampal Injection of ANA-12 in Rats

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The female rats were anesthetized with 4% isoflurane in oxygen. The skull was exposed, and the head was positioned in a stereotaxic apparatus. A 10-μL Hamilton syringe (Hamilton, Bonaduz AG, Bonaduz, Switzerland) with a stainless needle was used to inject 0.1 pmol/1 μL ANA-12 (Tocris, Bristol, UK, pH 7.4 adjusted with NaOH) or aCSF into the hippocampus with an infusion pump (UMC4; World Precision Instruments, FL, USA) over the course of 2 min. The needle was left in place for another 5 min before removal, and the total procedure lasted 7 min. The coordinates for injection were as follows: anterior/posterior (AP) −3.8 mm, lateral (L) +/−2.0 mm, and dorsal/ventral (DV) −2.5 mm^{59 (link)}. The correct position of the needle was assessed by brain slide capture (Fig. 7a). The ANA-12 dose and administration method were based on a previous work^{60 (link)}. Postoperatively, the rats remained in a warm cage until they recovered.

Seo S.Y., Moon J.Y., Kang S.Y., Kwon O.S., Kwon S., Bang S.K., Kim S.P., Choi K.H, & Ryu Y. (2018). An estradiol-independent BDNF-NPY cascade is involved in the antidepressant effect of mechanical acupuncture instruments in ovariectomized rats. Scientific Reports, 8, 5849.

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Diabetes and Cardiac Biomarkers in Mice

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For the present study, male db/db mice and male C57BL/6 mice were employed. The animals were maintained on the standard diet and these had free access to drinking water. The animals were kept in the polypropylene cages in the group of three (maximum) in the animal house at a temperature of 24°–26° with relative humidity of 40%–60%. The animals were exposed to 12 h light and dark cycle. The floor of the cages was covered with husk and each cage was cleaned completely every week. The Institutional Animal Care and Use Committee of Cadre Ward 901 Hospital of the Joint Logistics Support Unit of the Chinese People's Liberation Army approved the animal ethical protocol (Ethical approval number: 202000876A). All experiments related to animals were performed as per Institutional and International ethical guidelines. The kits for estimating the levels of creatine kinase (CK-MB), lactate dehydrogenase-1 (LDH-1) and cardiac troponin T (cTnT) were purchased from Jiancheng Reagent Co, Nanjing, China. The ELISA kits were employed for the quantification of BDNF, p-GSK-3β/GSK-3β and Nrf-2 (MyBioSource, Inc., San Diego, CA, USA). ANA-12 was employed as a specific BDNF receptor antagonist (Tocris Biosciences, Minneapolis, MN, USA). The doses of L-NAME [22 (link)] and ANA-12 [23 (link)] were used as per literature reports.

Li Y., Huang Y., Cheng X., He Y, & Hu X. (2021). Whole body hypoxic preconditioning-mediated multiorgan protection in db/db mice via nitric oxide-BDNF-GSK-3β-Nrf2 signaling pathway. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 25(4), 281-296.

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Neurotrophin and Receptor Antagonism Study

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Cultured cells were incubated for 24 h with 40 μM NTS (Calbiochem/Merck Millipore, Fontenay sous Bois, France) or 100 ng/ml human recombinant BDNF (Alomone labs, Jerusalem, Israel). Optimal concentrations of exogenous BDNF and NTS were determined previously.^{14 (link), 21 (link)} The non-peptide NTSR antagonist SR142948A (Tocris Biosciences, Bristol, UK) was used at 67 nM,^{56 (link)} PTX (Sigma-Aldrich, St Louis, MO, USA) at 200 ng/ml^{35 (link), 57 (link)}; TrkB inhibitors were K252a (Alomone Labs) at 100 nM^{58 (link), 59 (link)} or ANA12 (Tocris Biosciences) at 100 μM.^{60 (link)}

Abbaci A., Talbot H., Saada S., Gachard N., Abraham J., Jaccard A., Bordessoule D., Fauchais A.L., Naves T, & Jauberteau M.O. (2017). Neurotensin receptor type 2 protects B-cell chronic lymphocytic leukemia cells from apoptosis. Oncogene, 37(6), 756-767.

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