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Ptp1b

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PTP1B is a protein tyrosine phosphatase enzyme that plays a key role in the regulation of cellular signaling pathways. It functions as a negative regulator of insulin and leptin signaling, making it an important target for research in the areas of metabolic disorders and obesity.

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Lab products found in correlation

Opti mem, by Thermo Fisher Scientific (4 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (4 mentions) E cadherin, by Cell Signaling Technology (2 mentions) Vinculin, by Santa Cruz Biotechnology (2 mentions) β actin, by Merck Group (2 mentions) Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions) Sr321721 c, by Merck Group (2 mentions) Veptp, by Merck Group (2 mentions) Sasi hs02 00324728, by Merck Group (2 mentions) Si0265877 hs ptprj 6, by Qiagen (2 mentions) α glucosidase, by Merck Group (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions) Amersham hyperfilm, by GE Healthcare (1 mentions) Mouse anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions) Ecl reagent, by GE Healthcare (1 mentions) Phosphatidylinositol pi, by Avanti Polar Lipids (1 mentions) Pvdf membrane, by Bio-Rad (1 mentions) Stat3, by Merck Group (1 mentions) Stat5, by Merck Group (1 mentions)

15 protocols using ptp1b

1

PTP1B Inhibition Assay Protocol

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Extracts of A. cominia at 30 µg/ml were incubated according to the method described by Montalibet et al. (2005) with Protein Tyrosine Phosphatase 1B (PTP 1B, purchased from Invitrogen, Life Technologies, UK) at concentrations of 2 nM for 30 min at 37 °C in a humidified atmosphere containing 5% CO 2 . 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP, 10 µM, Invitrogen D6567, Molecular Probes, Life Technologies, UK) was then added and incubated for 10 min at 37 °C. TFMS [Bis (4-trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzine, Calbiochem 540211, 10 mg, Millipore, UK] was used as a standard inhibitor for the PTP1B inhibition assay in a concentration range of 0.0003-3.00 µM. The hydrolysis of DiFMUP by PTP1B enzyme was measured at λ max 355 nm (extinction) and 460 nm (emission).
Semaan D.G., Igoli J.O., Young L., Marrero E., Gray A.I, & Rowan E.G. (2017). In vitro anti-diabetic activity of flavonoids and pheophytins from Allophylus cominia Sw . on PTP1B, DPPIV, alpha-glucosidase and alpha-amylase enzymes. Journal of ethnopharmacology, 203.
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2

Signaling Cascade Analysis in RK-682 Treated RH-7777 Cells

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McArdle RH-7777 cells were obtained from the American Type Culture Collection (ATCC® CRL-1601™, Manassas, VA). Materials and reagents were essentially as described previously [10 (link)]. RK-682 was obtained from Enzo Life Sciences, Inc. (Farmingdale, NY). Phosphatase inhibitor cocktail II and protease inhibitor cocktail III were obtained from Calbiochem (San Diego, CA). PROTEAN®TGX™ (TGX) SDS polyacrylamide gels (4–15% acrylamide) and PVDF membranes were obtained from Bio-Rad Laboratories (Hercules, CA). Mouse anti-pY monoclonal antibody (4G10) and rabbit anti-IRS1, IRS2, PTP1B and p85 were from Millipore (Temecula, CA). Mouse anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-mouse and anti-rabbit horseradish peroxidase (HRP)-linked IgG, ECL reagents, and Amersham™ Hyperfilm™ were from GE Healthcare (Buckinghamshire, UK). For PI3K assays, substrate phosphatidylinositol (PI) was obtained from Avanti Polar Lipids (Alabaster, AL), and [γ-32P]ATP (3000 Ci/mmol) was from Perkin Elmer-NEN Life Science Products (Waltham, MA).
Sparks J.D., Magra A.L., Chamberlain J.M., O’Dell C, & Sparks C.E. (2015). Insulin dependent apolipoprotein B degradation and phosphatidylinositide 3-kinase activation with microsomal translocation are restored in McArdle RH7777 cells following serum deprivation. Biochemical and biophysical research communications, 469(2), 326-331.
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3

Western Blot Analysis of Signaling Proteins

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Tissues and cells were homogenized in radioimmunoprecipitation-assay buffer containing sodium-orthovanadate and protease inhibitors (9 (link)). Proteins were separated by SDS-PAGE (4-12%) and transferred to nitrocellulose. Immunoblotting was performed using antibodies from Cell Signalling Technologies, unless otherwise stated: p-STAT3 Y705, p-STAT3 S727, STAT3, p-P38 T108/Y182, P38, p-ERK T202/204, ERK, GAPDH, pSTAT5 Y694, STAT5 and PTP1B (Millipore). Immunoblots were visualized using enhanced chemilluminescence, and quantified using Bio-1D densitometry scanning software (PeqLab, UK).
Le Sommer S., Morrice N., Pesaresi M., Thompson D., Vickers M.A., Murray G.I., Mody N., Neel B.G., Bence K.K., Wilson H.M, & Delibegovic M. (2017). Deficiency in protein tyrosine phosphatase PTP1B shortens lifespan and leads to development of acute leukemia. Cancer research, 78(1), 75-87.
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4

Protein Expression Analysis in Liver Lysates

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Frozen liver lysates were prepared in RIPA buffer as described previously [27 (link)]. Proteins were separated by 4–12% SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were performed using antibodies from Cell Signaling Technology (Cell Signaling by NEB, Hitchin, UK) (unless stated otherwise) against PTP1B (Millipore, Watford, UK); phospho-S6 (s235/236); total S6; phospho-Akt/PKB (s473); phospho-IR (tyr1163/1163) (Invitrogen, Paisley, UK); IR-β (Santa Cruz, Dallas, TX, USA); total Akt/PKB (Santa Cruz, Dallas, TX, USA) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz, Dallas, TX, USA). Proteins were visualized using enhanced chemiluminescence and quantified by densitometry scanning (Image J) or Bio-1D software (Peqlab, Sarisbury Green, UK).
Lees E.K., Krol E., Shearer K., Mody N., Gettys T.W, & Delibegović M. (2014). Effects of hepatic protein tyrosine phosphatase 1B and methionine restriction on hepatic and whole-body glucose and lipid metabolism in mice. Metabolism: clinical and experimental, 64(2), 305-314.
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5

Inhibitor-based Signaling Pathway Analysis

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Growth factors were purchased from Life Technologies, Carlsbad, CA (EGF), and PeproTech, Rocky HIll, NJ (HGF, NRG-1, and IGF). The compounds used were Met inhibitor SU11274 (Selleck Chemicals, Houston, TX), EGFR inhibitor erlotinib (LC Labs, Woburn, MA; PeproTech), PTP1B inhibitor (539741; Calbiochem, Billerica, MA), and SHIP2 inhibitor AS1949490 (Tocris, Minneapolis, MN). The following antibodies were used: EGFR (Cell Signaling Technology, Danvers, MA), pEGFR Y1173 (Cell Signaling Technology; Epitomics), vimentin (BD Transduction Laboratories, San Jose, CA), E-cadherin (Cell Signaling Technology), PTP1B (Millipore, Burlington, MA), SHIP2 (Cell Signaling Technology), and panMena (Lebrand et al., 2004 (link)).
Hughes S.K., Oudin M.J., Tadros J., Neil J., Del Rosario A., Joughin B.A., Ritsma L., Wyckoff J., Vasile E., Eddy R., Philippar U., Lussiez A., Condeelis J.S., van Rheenen J., White F., Lauffenburger D.A, & Gertler F.B. (2015). PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena. Molecular Biology of the Cell, 26(21), 3867-3878.
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6

Quantitative Protein Expression Analysis

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Proteins were separated in 8–10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare). After electrotransfer, membranes were incubated for 1 h with low-fat milk powder (5%) in PBS containing 0.1% Tween-20. Blots were subsequently incubated with the appropriate antibodies against β-actin (A5441, Sigma), α-tubulin (T5168, Sigma), vinculin (sc-73614, Santa Cruz Biotechnology) as loading controls and α-SMA (A2547, Sigma), Type I Collagen (COL1A1) (234167, Calbiochem), Bcl-xL (610211, BD Biosciences), PTP1B (07–088, Merck-Millipore), cleaved caspase-3 (9661, Cell Signaling Technology), phospho-JNK1/2 (4668, Cell Signaling Technology), phospho-p38 MAPK (9211, Cell Signaling Technology), phospho-PDGFR (3161, Cell Signaling Technology) and phospho-AKT (Ser473) (sc-7985-R, Santa Cruz Biotechnology) at the dilutions recommended by the suppliers. Signals were detected using the ECL Western Blotting Detection Reagent (Bio-Rad). Values of densitometry were determined using the Quantity One software (Bio-Rad).
García-Ruiz I.G., Ruiz N.B., Rada P., Pardo V., Ruiz L., Blas-García A., Valdecantos M.P., Sanz M.G., Solís Herruzo J.A, & Valverde Á.M. (2019). Protein tyrosine phosphatase 1b deficiency protects against hepatic fibrosis by modulating nadph oxidases. Redox Biology, 26, 101263.
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7

Immunoblotting Reagent Suppliers

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All chemicals were purchased from Merck (Darmstadt, Germany) unless otherwise noted. Specific antibodies against actin were from Thermo Fisher Scientific (Waltham, MA, USA), PTP1B from Merck, GHR and phosphorylated (p) ATP citrate lyase (pACL) from Cell Signaling Technology (Danvers, MA, USA), pACC, FASN, ObRb, vinculin and sst2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and SOCS3 from Proteintech Europe (Manchester, UK). The Immun-Star Western C kit was from Bio-Rad Laboratories (Hercules, CA, USA). The corresponding secondary antibodies conjugated with horseradish-peroxidase, the high-capacity cDNA kit and TaqMan gene expression assays were purchased from Thermo Fisher Scientific.
Barrios V., Frago L.M., Canelles S., Guerra-Cantera S., Arilla-Ferreiro E., Chowen J.A, & Argente J. (2021). Leptin Modulates the Response of Brown Adipose Tissue to Negative Energy Balance: Implication of the GH/IGF-I Axis. International Journal of Molecular Sciences, 22(6), 2827.
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8

Knockdown of Signaling Regulators in HUVEC

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HUVEC were seeded onto 6-well plates and transfected at 80% confluency with 2.5 ml Opti-MEM (Thermo Fisher) with indicated siRNA plus 2.5 μL Lipofectamine RNA iMAX (Thermo Fisher) for 6–8 hrs. Transfection mix was replaced with full media (EGM-2 MV) for 48 hours, then cell starved in 2% FBS for 6–8 hrs before growth factor stimulation. Following siRNA were used for knockdown experiments in HUVEC: Sdc2 (Origene #SR321721 – C, 40 nM), VEPTP (Sigma-Aldrich SASI_Hs02_00324728, 60 nM), PTP1B Sigma-Aldrich SAS1_Hs01_00230699, 60nM) DEP1 (Qiagen #SI0265877 HS_PTPRJ_6, 60 nM)
Corti F., Ristori E., Rivera-Molina F., Toomre D., Zhang J., Mihailovic J., Zhuang Z, & Simons M. (2022). Syndecan-2 selectively regulates VEGF-induced vascular permeability. Nature cardiovascular research, 1(5), 518-528.
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9

Inhibition assay of PTPRZ1 and PTP1B

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Human recombinant PTPRZ1 and PTP1B proteins were purchased from Sigma (Madrid, Spain). The inhibitory activities of the studied compounds were determined using the phosphate sensor reagent (Thermofisher, Waltham, MA, USA) [46 (link)], using p-NPP (Sigma-Aldrich, Madrid, Spain) as substrate. PTPRZ1 and PTP1B were used at a concentration of 0.1 μM. Increasing concentrations of the different compounds were used (0.001–100 μM) to calculate the IC50. In each experiment, the hydrolysis of the p-NPP residue was determined as an increase in fluorescence at 450 nm (excitation at 430 nm) using a Hitachi F4500 Fluorescence Spectrometer.
Pastor M., Fernández-Calle R., Di Geronimo B., Vicente-Rodríguez M., Zapico J.M., Gramage E., Coderch C., Pérez-García C., Lasek A.W., Puchades-Carrasco L., Pineda-Lucena A., de Pascual-Teresa B., Herradón G, & Ramos A. (2017). Development of Inhibitors of Receptor Protein Tyrosine Phosphatase β/ζ (PTPRZ1) as Candidates for CNS disorders. European journal of medicinal chemistry, 144, 318-329.
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10

Knockdown of Signaling Regulators in HUVEC

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HUVEC were seeded onto 6-well plates and transfected at 80% confluency with 2.5 ml Opti-MEM (Thermo Fisher) with indicated siRNA plus 2.5 μL Lipofectamine RNA iMAX (Thermo Fisher) for 6–8 hrs. Transfection mix was replaced with full media (EGM-2 MV) for 48 hours, then cell starved in 2% FBS for 6–8 hrs before growth factor stimulation. Following siRNA were used for knockdown experiments in HUVEC: Sdc2 (Origene #SR321721 – C, 40 nM), VEPTP (Sigma-Aldrich SASI_Hs02_00324728, 60 nM), PTP1B Sigma-Aldrich SAS1_Hs01_00230699, 60nM) DEP1 (Qiagen #SI0265877 HS_PTPRJ_6, 60 nM)
Corti F., Ristori E., Rivera-Molina F., Toomre D., Zhang J., Mihailovic J., Zhuang Z, & Simons M. (2022). Syndecan-2 selectively regulates VEGF-induced vascular permeability. Nature cardiovascular research, 1(5), 518-528.
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