Phospho mypt1

Manufactured by Cell Signaling Technology

Sourced in United States

Phospho-MYPT1 is a product that detects the phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) protein. MYPT1 is a regulatory subunit of the myosin light chain phosphatase (MLCP) complex, which plays a role in the regulation of smooth muscle contraction. The Phospho-MYPT1 product can be used to measure the phosphorylation state of MYPT1, which is an indicator of MLCP activity.

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Lab products found in correlation

Acetone, by Thermo Fisher Scientific (3 mentions) Phorbol 12 13 dibutyrate, by Merck Group (3 mentions) Dithiothreitol (dtt), by Thermo Fisher Scientific (3 mentions) U46619, by Merck Group (3 mentions) Potassium chloride (kcl), by Merck Group (3 mentions) Acetylcholine, by Merck Group (3 mentions) Sodium fluoride, by Merck Group (3 mentions) Phospho mlc, by Cell Signaling Technology (2 mentions) Phospho ampk, by Cell Signaling Technology (2 mentions) Mypt1, by Cell Signaling Technology (2 mentions) Anti rhoa, by Cell Signaling Technology (1 mentions) Anti mlc, by Cell Signaling Technology (1 mentions) Anti ampk, by Abcam (1 mentions) Anti mypt1, by Abcam (1 mentions) Anti o glcnac, by Thermo Fisher Scientific (1 mentions) Mlc20 antibody, by Merck Group (1 mentions) Y 27632, by Merck Group (1 mentions) Phenylephrine, by Merck Group (1 mentions) Pde4d, by Abcam (1 mentions) Pde4b, by Cell Signaling Technology (1 mentions)

9 protocols using phospho mypt1

Aortic Protein Expression Analysis

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Proteins (60 µg) extracted from aortas or VSMCs were separated by electrophoresis on
10% polyacrylamide gels and transferred to nitrocellulose membranes. Nonspecific
binding sites were blocked with 5% skim milk in Tris-buffered saline solution with
10% Tween for 1 h at 24°C. Membranes were then incubated with antibodies overnight at
4°C. Anti-O-GlcNAc (CTD 110.6, 1:2000; Pierce Biotechnology, USA), anti-AMPK (#80039,
1:1000; Abcam, USA), anti-protein kinase CPI-17 (#32213, 1:1000; Abcam, USA),
anti-MYPT-1 (#2634), anti-rho-kinase (ROCK)-α (#8236), anti-ROCK-β (#4035), anti-MLC
(#8505) and anti-RhoA (#2117) (all 1:1000; Cell Signaling, USA, or BD Biosciences
Transduction Laboratories, USA) were used. Immunoblots for nonphosphoproteins were
carried out on the same membranes used to evaluate the phosphorylated (phospho-)
forms: phospho-MYPT-1 (Thr⁸⁵³), phospho-CPI-17 (Thr³⁸),
phospho-MLC (Thr¹⁸/Ser¹⁹), and phospho-AMPK
(Thr¹⁷²), (1:500; Cell Signaling, USA). After incubation with secondary
antibodies, signals were developed for chemiluminescence, visualized by
autoradiography, and quantified densitometrically. Results were normalized to
beta-actin protein (#A5316, 1:10000; Sigma-Aldrich, Inc., USA), or to the total form
of each phosphorylated protein, and reported as arbitrary units.

Lima V.V., Lobato N.S., Filgueira F.P., Webb R.C., Tostes R.C, & Giachini F.R. (2014). Vascular O-GlcNAcylation augments reactivity to constrictor stimuli by prolonging phosphorylated levels of the myosin light chain. Brazilian Journal of Medical and Biological Research, 47(10), 826-833.

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Assessing RhoA/Rho-kinase and MEK Activity

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Sodium fluoride, KCl, phenylephrine, acetylcholine, U46619, Y-27632, and phorbol 12,13-dibutyrate were purchased from Sigma (St. Louis, MO, USA). DTT, TCA, and acetone were obtained from Fisher Scientific (Hampton, NH, USA). Enhanced chemiluminescence (ECL) kits were from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase targeting subunit protein 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, ERK, or phosphoERK at Thr202/Tyr204 (Cell Signaling Technology, Danvers, MA, USA or Upstate Biotechnology, Lake Placid, NY, USA) were used to determine levels of RhoA/Rho-kinase activity (Wooldridge et al., 2004 (link); Wilson et al., 2005 (link)) or MEK activity. Anti-mouse IgM (goat) and anti-rabbit IgG (goat) conjugated with horseradish peroxidase were used as secondary antibodies (1:2,000 dilutions for both, Upstate). A specific MLC₂₀ antibody (1:1500, Sigma) and anti-mouse IgG (goat) conjugated to horseradish peroxidase (1:2000, Upstate) were used to determine the level of LC₂₀ phosphorylation.

Chung Y.H., Oh K.W., Kim S.T., Park E.S., Je H.D., Yoon H.J., Sohn U.D., Jeong J.H, & La H.O. (2017). Hypothermia Inhibits Endothelium-Independent Vascular Contractility via Rho-kinase Inhibition. Biomolecules & Therapeutics, 26(2), 139-145.

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Western Blot Analysis of Aortic Tissues

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Protein was extracted from the aortic tissues or RASMCs in a lysis buffer. Equal quantities of protein extract (30 μg per lane) were separated by 8, 10, or 12% SDS–PAGE and transferred to a polyvinylidene fluoride membrane (Merck, Cat#: IPVH00010). The target protein was probed with numerous antibodies: PDE4A (1:1000, Thermo Fisher, Cat#:PA5-115730), PDE4B (1:1000, Cell Signaling Technology, Cat#: 72096S), PDE4C (1:1000, Thermo Fisher, Cat#: PA5-106624), PDE4D (1:1000, Abcam, Cat#: ab171750), AMP-activated protein kinase (AMPK; 1:1000, Cell Signaling Technology, Cat#: 2532S), Phospho-AMPK (1:1000, Cell Signaling Technology, Cat#: 2535S), myosin phosphatase targeting subunit 1 (MYPT1; 1:1000, Cell Signaling Technology, Cat#: 2634S), Phospho-MYPT1 (1:500, Cell Signaling Technology, Cat#: 5163S), MLC (1:1000, Cell Signaling Technology, Cat#: 8505S), and Phospho-MLC (1:1000, Cell Signaling Technology, Cat#: 3675S), respectively. Immunoblotting of the housekeeping protein GAPDH (1:5000, Proteintech, Cat#: 60004-1-Ig) was performed to ensure equal protein loading. Immunoreactive bands were visualized with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Pierce, Cat#: 34577). The protein expression was measured by analyzing the relative protein band intensity with Image-Pro Plus 6.0 software.

Fan T., Hou Y., Ge W., Fan T., Feng X., Guo W., Song X., Gao R, & Wang J. (2022). Phosphodiesterase 4D promotes angiotensin II-induced hypertension in mice via smooth muscle cell contraction. Communications Biology, 5, 81.

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C2C12 Myoblast Signaling Assay

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C2C12 mouse myoblasts were cultured in Dulbecco’s modified Eagle medium (DMEM) (Mediatech, Manassas VA) containing 10% fetal bovine serum (Hyclone, Logan, UT), and maintained at 37°C in a humidified 5% CO₂ atmosphere. Cells treated with or without meglumine at the concentrations and for the times indicated were harvested by scraping, washing twice in PBS and lysing in RIPA buffer containing protease and phosphatase inhibitors. Equal protein for each sample (typically 50 µg/lane) was separated by SDS–PAGE and transferred to Immobilon-P membranes (Millipore, Billerica MA). Blots were incubated with primary antibodies as recommended by the vendor (dilution 1∶1000) and were detected with HRP conjugated secondary antibodies using the enhanced chemilumescent reagents (Pierce, Rockford IL) according to the manufacturer’s instructions. Primary antibodies used were for SNARK (also known as NUAK2), MYPT1, phospho-MYPT1 (Ser507), phospho-MYPT1 (Ser668), phospho-MYPT1 (Thr696), (Cell Signaling Technology, Beverly, MA), Glut1 (Abcam, Cambridge, MA), AMPK (Millipore, Temecula, CA) and ß-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Bravo-Nuevo A., Marcy A., Huang M., Kappler F., Mulgrew J., Laury-Kleintop L., Reichman M., Tobia A, & Prendergast G.C. (2014). Meglumine Exerts Protective Effects against Features of Metabolic Syndrome and Type II Diabetes. PLoS ONE, 9(2), e90031.

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Rho-kinase Pathway Regulation Assay

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Drugs and chemicals were obtained from the following sources. Sodium fluoride, KCl, acetylcholine, apigenin, U46619 and phorbol 12,13-dibutyrate were purchased from Sigma (St. Louis, MO, USA). DTT, TCA and acetone were obtained from Fisher Scientific (Hampton, NH, USA). Enhanced chemiluminescence (ECL) kits were from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase targeting subunit protein 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, ERK or phosphoERK at Thr202/Tyr204 were purchased from Cell Signaling Technology (Danvers, MA, USA) or Upstate Biotechnology (Lake Placid, NY, USA) to determine levels of RhoA/Rho-kinase activity (Wilson et al., 2005 (link); Wooldridge et al., 2004 (link)) or MEK activity. Anti-mouse IgM (goat) and anti-rabbit IgG (goat), conjugated with horseradish peroxidase, were used as secondary antibodies (1:2,000 and 1:2,000, respectively, Upstate, Lake Placid, NY). apigenin was prepared in dimethyl sulfoxide (DMSO) as a 100 mM stock solution and frozen at −20°C for later use. DMSO alone had no observable effect at concentrations used (data not shown).

Je H.D., Kim H.D, & La H.O. (2014). The Inhibitory Effect of Apigenin on the Agonist-Induced Regulation of Vascular Contractility via Calcium Desensitization-Related Pathways. Biomolecules & Therapeutics, 22(2), 100-105.

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Rho-kinase Activity Assay Protocol

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Drugs and chemicals were obtained from the following sources. Sodium fluoride, KCl, acetylcholine, fisetin, U-46619 and phorbol 12,13-dibutyrate were purchased from Sigma (St. Louis, MO, USA). DTT, TCA and acetone were obtained from Fisher Scientific (Hampton, NH, USA). Enhanced chemiluminescence (ECL) kits were from Pierce (Rockford, IL, USA). Antibodies against phospho-myosin phosphatase targeting subunit protein 1 (phospho-MYPT1) at Thr855 (1:5,000), MYPT1, ERK or phosphoERK at Thr202/Tyr204 were purchased from Cell Signaling Technology (Danvers, MA, USA) or Upstate Biotechnology (Lake Placid, NY, USA) to determine levels of RhoA/Rho-kinase activity (Wooldridge et al., 2004 (link); Wilson et al, 2005 (link)) or MEK activity. Anti-mouse IgM (goat) and anti-rabbit IgG (goat), conjugated with horseradish peroxidase, were used as secondary antibodies (1:2,000 and 1:2,000, respectively, Upstate, Lake Placid, NY). fisetin solution was prepared in dimethyl sulfoxide (DMSO) as a 100 mM stock solution and frozen at −20°C for later use. DMSO alone had no observable effect at concentrations used (data not shown).

Je H.D., Sohn U.D, & La H.O. (2016). Endothelium-Independent Effect of Fisetin on the Agonist-Induced Regulation of Vascular Contractility. Biomolecules & Therapeutics, 24(1), 57-61.

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Leptin Signaling Pathway Analysis

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After treatment with leptin or the control, the cells were harvested in cold PBS, and the pellet was re-suspended in lysis buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol) for 20 min at 4ºC. Next, the lysate was sonicated and centrifuged at 14,000 g for 20 min at 4°C, after which, the supernatant was collected. The protein concentrations were determined using the Bradford assay. A total of 50-100 μg of the protein extract was loaded in each lane, separated on a 10% SDS-PAGE gel, transferred to nitrocellulose membranes and incubated overnight with the following primary antibodies: leptin receptor (1:1000) and CD44 (1:1000) (Santa Cruz Biotechnology, CA, USA); phospho-STAT3 (1:1000), MAPK (1:1000), phospho-MAPK (1:1000), phospho-AKT (1:1000), mTOR (1:1000), phospho-MYPT1 (1:1000), RhoA (1:1000), PARP (1:1000), and Snail (1:1000) (Cell Signaling Technology), Nanog (1:1000) (R&D Systems); and N-cadherin (1:1000), E-cadherin (1:500), Oct-4 (1:1000), Nanog (1:1000), Zeb2 (1:1000), Vimentin (1:1000) (Abcam) and β-actin (1:10000) (Sigma-Aldrich Corp.). Peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibodies (1:3000, Bio-Rad Labs, CA, USA) were applied for one hour at room temperature (RT). The reaction was developed with chemiluminescence using the ECL Western blot analysis system (NEN, Western lightning, Perkin-Elmer).

Kato S., Abarzua-Catalan L., Trigo C., Delpiano A., Sanhueza C., García K., Ibañez C., Hormazábal K., Diaz D., Brañes J., Castellón E., Bravo E., Owen G, & Cuello M. (2015). Leptin stimulates migration and invasion and maintains cancer stem-like properties in ovarian cancer cells: an explanation for poor outcomes in obese women. Oncotarget, 6(25), 21100-21119.

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Phosphorylation of MYPT1 in PAH-SMCs

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PAH-SMCs were cultured in SMC growth medium. Before the treatment, PAH-SMCs were starved for 21 h in basal medium supplemented with 1% antibiotics. Serum-starved PAH-SMCs were then treated with 1 μM of fasudil or FasPRO for 24 h, either under normoxic or hypoxic conditions. Whole-cell lysates from the treated cells were collected using lysis buffer [20 mM Tris-HCl (pH 7.4), 15% glycerol, 1% Triton X-100, 8 mM MgSO₄, 150 mM NaCl, 1 mM EDTA, supplemented with β-glycerophosphate, NaF, protease inhibitor, and phosphatase inhibitor]. After centrifuging (14000 x g for 10 min at 4ºC) the whole-cell lysates, we determined the protein concentration using a BCA protein assay kit (Pierce Biotechnology, IL). The cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin and incubated with primary antibodies against phospho-MYPT1, MYPT1, and β-Actin (Cell Signaling, MA) followed by incubating with goat anti-rabbit and goat anti-mouse IgG HRP-conjugated secondary antibodies. Immunoreactivity of the proteins was visualized using a ChemiDoc™ XRS molecular imager system (Bio-Rad, CA) after incubating the membranes with WesternSure^® chemiluminescence reagent (LI-COR Biosciences, NE) for 1 min.

Al-Hilal T.A., Hossain M.A., Alobaida A., Alam F., Keshavarz A., Nozik-Grayck E., Stenmark K.R., German N.A, & Ahsan F. (2021). Design, synthesis and biological evaluations of a long-acting, hypoxia-activated prodrug of fasudil, a ROCK inhibitor, to reduce its systemic side-effects. Journal of controlled release : official journal of the Controlled Release Society, 334, 237-247.

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Cellular Signaling Pathway Analysis

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All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless indicated otherwise. Antibodies were from Cell Signaling Technology, Inc. (Danvers, MA): Epac1, Vav2, Rap1a, Rap1b, Tiam, diphospho-MLC (Thr18/Ser19) and phospho-MYPT1 (Thr853). All antibodies are rabbit monoclonal antibodies. HRP-linked anti-actin, and GAPDH were rabbit monoclonal antibodies. ECIS arrays (8W10E+) were from Applied BioPhysics (Troy, NY). Rac1 G-LISA Activation Assay Kit was purchased from Cytoskeleton Inc. (Denver, CO). LacZ adenoviral control vector was obtain from Invitrogen (Carlsbad, CA).

Kovacs-Kasa A., Kim K.M., Cherian-Shaw M., Black S.M., Fulton D.J, & Verin A.D. (2018). Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: molecular mechanisms and barrier-protective role. Journal of cellular physiology, 233(8), 5736-5746.

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