Kt5720

Manufactured by Enzo Life Sciences

Sourced in United States, Germany

KT5720 is a potent and selective inhibitor of the cAMP-dependent protein kinase (PKA). It blocks the ATP-binding site of the PKA catalytic subunit, thereby inhibiting its enzymatic activity. KT5720 is a commonly used tool in biomedical research to study the role of PKA signaling in various cellular processes.

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5 protocols using kt5720

Biochemical Characterization of PKA Signaling

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HEK293T, HEK293 β2AR [36 (link)] cells and U87-MG cells (ATCC^® HTB-14) were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The colon cancer cell lines SW480 (ATCC^® CCL-228) and SW620 (ATCC^® CCL-227) were grown in RPMI-1640 media supplemented with 10% FBS. Cells were grown in water-saturated, 5% CO₂ atmosphere. Transient transfections were performed with Transfectin reagent (Bio-Rad, #1703352). Forskolin was purchased from MCE Med Chem Express (#HY-15371), Isoproterenol was purchased from Sigma (#I6504), KT5720 was purchase from Enzo Life Sciences (BML-EI199-0100), and employed with indicated concentrations and time frames. Primary antibodies used were the mouse anti-GFP antibody (Roche, #11814460001, Germany), the rabbit polyclonal Phospho-PKA Substrate (RRXS*/T*) antibody (Cell Signaling, #9624), the rabbit polyclonal TAF15 antibody (Cell Signaling, #13150), the mouse monoclonal (mAb) Anti- PKA RIα (D54D9) antibody (BD Biosciences, #610166), and the mouse monoclonal Anti-PKAc antibody (BD Biosciences, #610981). Lamin A/C (Cell Signaling, #4777) Mouse mAb, GAPDH (Cell Signaling, #2118) Rabbit mAb, Phospho-VASP (Ser157) (Cell Signaling, #84519) Rabbit mAb.

Feichtner A., Enzler F., Kugler V., Hoppe K., Mair S., Kremser L., Lindner H., Huber R.G., Stelzl U., Stefan E, & Torres-Quesada O. (2024). Phosphorylation of the compartmentalized PKA substrate TAF15 regulates RNA–protein interactions. Cellular and Molecular Life Sciences: CMLS, 81(1), 162.

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Alzheimer's Protectant Compound Characterization

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Cilostazol [OPC-13013, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3,4-dihydro-2-(1H)-quinolinone] was donated by Otsuka Pharmaceutical Co. Ltd. (Tokushima, Japan) and dissolved in DMSO to prepare a 10 mM stock solution. Recombinant SIRT1, resveratrol, and bafilomycin A1 (Sigma-Aldrich) were dissolved in DMSO. Sirtinol (Calbiochem) was dissolved in DMSO (vehicle < 0.1% v/v on final volume). Aβ1–42 peptide (appearance, white powder; HPLC purity, >95%) was purchased from AnaSpec (catalog No.24236, run No. 77828; AnaSpec, Fremont, CA), dissolved in 1% NH₄OH (basic buffer) as a stock solution at 1 mM and stored at -20°C. When diluted, the stock solution of Aβ1–42 was clear, seedless, and showed no sign of aggregation. KT5720 (a PKA inhibitor) was purchased from Enzo Life Sciences, and MTT, 3-methyladenine, bafilomycin A1, and tetracycline were from Sigma-Aldrich. TIMP-1 (Calbiochem) was dissolved in phosphate-buffered saline.

Lee H.R., Shin H.K., Park S.Y., Kim H.Y., Bae S.S., Lee W.S., Rhim B.Y., Hong K.W, & Kim C.D. (2015). Cilostazol Upregulates Autophagy via SIRT1 Activation: Reducing Amyloid-β Peptide and APP-CTFβ Levels in Neuronal Cells. PLoS ONE, 10(8), e0134486.

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Sciatic Nerve PKA Inhibition Study

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Treatments with the PKA signaling inhibitors H-89 (EMD Millipore) and KT5720 from (Enzo Life Sciences) were performed through injections beneath the gluteus superficialis and biceps femoris muscles. Animals were injected on 4 consecutive days from P3 to P6 with 10 μl of inhibitor solution (10 μM H-89 DMSO, 1 μM KT5720, 0.1% DMSO diluted PBS) per day. Untreated nerves were injected with 10 μl of 0.1% DMSO diluted in PBS. Sciatic nerves were sampled at P7.

Ghidinelli M., Poitelon Y., Shin Y.K., Ameroso D., Williamson C., Ferri C., Pellegatta M., Espino K., Mogha A., Monk K., Podini P., Taveggia C., Nave K.A., Wrabetz L., Park H.T, & Feltri M.L. (2017). Laminin 211 inhibits protein kinase A in Schwann cells to modulate neuregulin 1 type III-driven myelination. PLoS Biology, 15(6), e2001408.

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Murine Pancreatic Acinar Cell Cultures

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266-6 cells, a murine pancreatic acinar cell line, were maintained in Dulbecco’s Modified Eagles Medium (DMEM; Hyclone; Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone). Culture plates were coated with 0.1% pig skin gelatin (Sigma, St. Louis, MO, USA) in phosphate-buffered saline (PBS) for 30 to 60 min prior to use. AR42J cells, a rat pancreatic acinar cell line, were maintained in F-12K Medium (Kaighn’s Modification of Ham’s F-12 Medium containing 2 mM L-glutamine and 1500 mg/L sodium bicarbonate; ATCC, Manassas, VA, USA) supplemented with 20% FBS. HEK293T cells were maintained in DMEM supplemented with 10% FBS. Primary pancreatic acinar cells were prepared from C57BL/6 mice as described previously [23 (link)]. To examine the role of β-AR and cAMP-dependent protein kinase (PKA) pathway, cells were treated with the following reagents: 1 μM isoproterenol (an agonist of β1-, β2- and β3-AR; Sigma), 10 μM propranolol, 10 nM acebutolol (β1-selective antagonist; Sigma), 10 nM ICI-118,551 (β2-selective antagonist; Sigma), 10 nM Rp-cAMPs (an inhibitor of PKA activation; Enzo Life Sciences; Farmingdale, NY, USA), 5 μM KT5720 (a specific inhibitor of PKA; Enzo Life Sciences) and 20 μM H89 (an inhibitor of PKA; Sigma).

Baek K., Park D., Hwang H.R., Kim S.G., Lee H, & Baek J.H. (2018). Blocking β1/β2-Adrenergic Signaling Reduces Dietary Fat Absorption by Suppressing Expression of Pancreatic Lipase in High Fat-Fed Mice. International Journal of Molecular Sciences, 19(3), 857.

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Evaluation of Cellular Signaling Modulators

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Cell culture reagents were obtained from Invitrogen (Darmstadt, Germany). The anti-pERK-1/2 (E-4) and the anti-Rap-1a (sc-1482) antiserum was from Santa Cruz (Heidelberg, Germany). The pCREB-Ser-133 antibody was purchased from cell signaling (Leiden, the Netherlands). The anti-tubulin (clone 6-11B-1) and the peroxidase-conjugated anti-mouse or anti-rabbit antibody, both raised in goat, from Bio-Rad (München, Germany). The firefly luciferase substrate was from Promega (Mannheim, Germany). rp-Br-cAMPs and γ-MSH were from SigmaAldrich (Deisenhofen, Germany). ESI-09, ESI-05 and HJC-0197 were from Biolog (Bremen, Germany), A-812511 from abcam (Cambridge, UK) and PD-184,352, HS-024 or NPY from Tocris (Bristol, UK). α-MSH was from Biotrend (Cologne, Germany) and KT-5720 from Enzo life science (Lörrach, Germany).

Glas E., Mückter H., Gudermann T, & Breit A. (2016). Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression. Scientific Reports, 6, 32776.

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