Ptp1b

Manufactured by Cell Signaling Technology

Sourced in United States

PTP1B is a protein tyrosine phosphatase enzyme that plays a role in the regulation of cellular signaling pathways. It is involved in the dephosphorylation of phosphorylated tyrosine residues on target proteins.

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4 protocols using ptp1b

FFPE Protein Extraction and Immunoblotting

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Proteins (50 μg) were extracted from FFPE tissues and separated as mentioned. Primary antibodies were incubated overnight at 4°C as follows: anti-Ephrin-A3 (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PTP1B (1:200 dilution; Abcam, Cambridge, UK), and anti-VEGF (1:200 dilution; Abcam, Cambridge, UK). Secondary antibodies were used as follows: anti-rabbit for Ephrin-A3, PTP1B, and VEGF (1:2,000 dilution, Cell Signaling Technology, Danvers, MA, USA). Beta-actin was used as a loading control.

Jung K.O., Youn H., Lee C.H., Kang K.W, & Chung J.K. (2016). Visualization of exosome-mediated miR-210 transfer from hypoxic tumor cells. Oncotarget, 8(6), 9899-9910.

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Protein Expression and Analysis by Western Blot

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Cells were disrupted using a M-PER Mammalian Protein Extraction Reagent kit (Thermo SCIENTIFIC) and total proteins were quantified using Bradford Assay. Total proteins (50ug) were used for western-blot analysis, and were loaded onto 10–12% SDS-PAGE (Mini-protean TGX Stain-free gels, BioRad, Hercules, California, USA) with prestained protein standards. Primary antibodies (mTOR, PTP1B, PTEN, TCTP, LC3B, p62, Caspase 3 and β-actin) were purchased from Cell Signaling Technology (Beverly, MA, USA) and anti-core antibody from Enzo Life Science (Postfach, Lausen, Switzerland). Proteins were detected by chemiluminescence, according to manufacturer’s instructions (WesternBright^™ ECL, Advansta, Menlo Park, California, USA). Image analysis and quantification was performed using ChemiDoc^™ MP Imaging System and ChemiDoc^™ XRS+ software (BioRad).

del Campo J.A., García-Valdecasas M., Gil-Gómez A., Rojas Á., Gallego P., Ampuero J., Gallego-Durán R., Pastor H., Grande L., Padillo F.J., Muntané J, & Romero-Gómez M. (2018). Simvastatin and metformin inhibit cell growth in hepatitis C virus infected cells via mTOR increasing PTEN and autophagy. PLoS ONE, 13(1), e0191805.

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Western Blot Analysis of Signaling Pathways

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Western blotting was performed, as described previously [9 (link)], with primary antibodies PTP1b (Cat # CST5311, Cell Signaling, 1:1000), BMPR2 (Cat # MA5-15827, Invitrogen, 1:800), pSMAD1/5/9 (Cat # 13820S, Cell Signaling, 1:1000), ID1 (Cat # sc-133104, Santa Cruz, 1:100), and b-Actin (Cat # SC4778, Santa Cruz, 1:600), and secondary antibodies goat anti-rabbit IgG H&L HRP (Cat # ab6721, Abcam, 1:5000) and goat anti-mouse IgG H&L HRP (Cat # ab205719, Abcam, 1:5000).

Ali M.K., Tian X., Zhao L., Schimmel K., Rhodes C.J., Wilkins M.R., Nicolls M.R, & Spiekerkoetter E.F. (2023). PTPN1 Deficiency Modulates BMPR2 Signaling and Induces Endothelial Dysfunction in Pulmonary Arterial Hypertension. Cells, 12(2), 316.

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Protein Extraction and Western Blotting

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For total protein extraction, the frozen liver tissue was homogenised in Nonidet P-40 lysis buffer. The following antibodies were used for western blotting: Nrf2 (sc-722), IL-1β (sc-7884), IL-6 (sc-7920) and BDNF (sc-546) (Santa Cruz Biotechnology, Dallas, TX); pIKK (#2697), STAT3 (#4904), FOXO1 (#2880), SOCS3 (#2932), and PTP1B (#5311) (Cell Signalling Technology, Beverly, MA). Both nuclear and cytosolic protein levels of Nrf2 were analysed. The bands corresponding to the proteins of interest were scanned and the band density analysed using the automatic imaging analysis system, Quantity One (Bio-Rad Laboratories, Hercules, California) as described in our previous study (Camer, Yu, Szabo et al., 2015) . All quantitative analyses for total and cytosolic proteins were normalised to β-actin.
Nuclear proteins were normalised to Lamin B.

Camer D., Yu Y., Szabo A., Dinh C.H., Wang H., Cheng L, & Huang X.F. (2015). Bardoxolone methyl prevents insulin resistance and the development of hepatic steatosis in mice fed a high-fat diet. Molecular and cellular endocrinology, 412.

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