Phospho stat5

Manufactured by Cell Signaling Technology

Sourced in United States, Germany

Phospho-STAT5 is a laboratory product from Cell Signaling Technology. It is used to detect and quantify the phosphorylated form of STAT5, a transcription factor that plays a critical role in signal transduction pathways.

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Lab products found in correlation

55 protocols using phospho stat5

Investigating FLT3 Signaling Pathways

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MOLM14, MV4-11 cells were treated with DMSO, serially diluted A674563, TCS359 (1 μM), MK2206 (1 μM) for 4 hours. Cells were then washed in PBS and lysed in cell lysis buffer. FLT3, Phospho-FLT3 (Tyr589/591), AKT, Phospho-AKT Ser473, Phospho-AKT Thr308, GSK-3β, Phospho-GSK-3β (Ser9), Phospho-FoxO1 (Thr24), FoxO1, PRAS40, Phospho-PRAS40 (Thr246), STAT5, Phospho-STAT5 (Tyr694), NF-ΚB-P65, Phospho-NF-ΚB-P65 (Ser536), P70S6K, Phospho-P70S6K Thr389, 4EBP1, Phospho-4EBP1 (Thr37/46), ERK, Phospho-p44/42MAPK (Erk1/2) (Thr202/Tyr204), C-Myc and GAPDH antibodies (Cell Signaling Technology) were used for immunoblotting.
For FL addition experiment, MV4-11 cells were treated with FLT3 inhibitors in the presence of 10 ng/mL of FL (FLT3 Ligand, R&D Systems) or absence of FL for 2 hrs. Cells were then washed in PBS and lysed in cell lysis buffer. FLT3, Phospho-FLT3 (Tyr589/591), AKT, GSK-3β, Phospho-GSK-3β (Ser9), Phospho-FoxO1 (Thr24), FoxO1, STAT5, Phospho-STAT5 (Tyr694), P70S6K, Phospho-P70S6K (Thr389), 4EBP1, Phospho-4EBP1 (Thr37/46), C-Myc and GAPDH antibodies (Cell Signaling Technology) were used for immunoblotting.

Wang A., Wu H., Chen C., Hu C., Qi Z., Wang W., Yu K., Liu X., Zou F., Zhao Z., Wu J., Liu J., Liu F., Wang L., Stone R.M., Galinksy I.A., Griffin J.D., Zhang S., Weisberg E.L., Liu J, & Liu Q. (2016). Dual inhibition of AKT/FLT3-ITD by A674563 overcomes FLT3 ligand-induced drug resistance in FLT3-ITD positive AML. Oncotarget, 7(20), 29131-29142.

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Immunoblot Analysis of Signaling Pathways

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Cells were lysed in RIPA buffer with added protease inhibitors and protein content was measured by BCA assay. Total protein was resolved using SDS-PAGE and subjected to immunoblot analysis. Nuclear and cytoplasmic protein fractions were separately processed similar to total protein and analyzed by immunoblot. Densitometric analyses were performed using ImageJ and phospho-p65 values were normalized to total p65. Uncropped blots are included with supplemental data. Antibodies used were phospho-STAT3 (1:1000, Cell Signaling #9131S), phospho-STAT5 (1:1000, Cell Signaling 9314S), phospho-p65 (1:1000, Cell Signaling #3033S), total STAT3 (1:1000, Cell Signaling #12640), total STAT5 (1:1000, Cell Signaling #94205), total p65 (1:1000, Cell Signaling #8242), GAPDH (1:10,000, Cell Signaling #2118), β-tubulin (1:1000, Cell Signaling #2146S), Lamin B1 (1:1000, Cell Signaling #12586S). We detected two bands for total STAT5 proteins, which correspond to Stat5a and STAT5b, isoforms of STAT5 encoded by homologous genes^{24 (link)}. We also detected two bands for p65, which may correspond to one of the reported isoforms for p65, of which the larger one is transcriptionally active^{25 (link)}.

Bapat A.S., O’Connor C.H, & Schwertfeger K.L. (2023). Targeting the NF-κB pathway enhances responsiveness of mammary tumors to JAK inhibitors. Scientific Reports, 13, 5349.

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Immunoblotting Analysis of Protein Targets

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Proteins were isolated using standard procedures. Whole-cell lysates containing 50 μg of proteins, were separated by SDS-PAGE and immunoblotted with the following specific antibodies: MYC, STAT5 and phospho-STAT5 (Cell Signaling Technologies, Danvers, MA, USA), ACTB (beta-actin) from (Sigma, St. Louis, MO, USA).

Ren M., Qin H., Wu Q., Savage N.M., George T.I, & Cowell J.K. (2016). Development of ZMYM2-FGFR1 driven AML in human CD34+ cells in immunocompromised mice. International journal of cancer, 139(4), 836-840.

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Dissecting PDGFR-mediated Signaling Pathways

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VSMCs were lysed in RIPA buffer and protein contents were measured using the BCA assay. Total lysates were immunoprecipitated with antibody against PDGFRβ (Santa Cruz Biotechnology, sc-432). Immunoprecipitates or cell lysates were subjected to Western blotting with antibodies to phospho-STAT1 (Invitrogen 333400), phospho-STAT3 (Cell Signaling, 9145), phospho-STAT5 (Cell Signaling, 9359), β-Actin (Cell Signaling, 4970L), phospho-Tyrosine (Millipore, 05–321), total STAT1 (Millipore, 06–501), total PLCγ1 (Cell Signaling 2822), and phosphor-PLCγ1 (Cell Signaling 14008). Primary antibodies were used at 1:1000. HRP-conjugated secondary antibodies (Jackson ImmunoResearch) were used at 1:5000.

He C., Medley S.C., Hu T., Hinsdale M.E., Lupu F., Virmani R, & Olson L.E. (2015). PDGFRβ signaling regulates local inflammation and synergizes with hypercholesterolemia to promote atherosclerosis. Nature communications, 6, 7770.

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FLT3 Phosphorylation and Signaling Assay

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Cells were treated with pacritinib or DMSO for 4 hours and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Immunoprecipitation was carried out overnight with anti-FLT3 antibodies (Santa Cruz Biotechnology, Dallas, TX). Dynabeads (ThermoFisher, Waltham, MA) were added the next day and incubated for 4 hours. Samples were then prepared according to manufacturer’s instructions. Eluted lysates or total cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes followed by western blot analysis using the indicated primary antibodies: FLT3, phospho-FLT3, STAT5, and phospho-STAT5 (Cell Signaling Technology, Danvers, MA).

Jeon J.Y., Zhao Q., Buelow D.R., Phelps M., Walker A.R., Mims A.S., Vasu S., Behbehani G., Blachly J., Blum W., Klisovic R.B., Byrd J.C., Garzon R., Baker S.D, & Bhatnagar B. (2019). Preclinical activity and a pilot phase I study of pacritinib, an oral JAK2/FLT3 inhibitor, and chemotherapy in FLT3-ITD-positive AML. Investigational new drugs, 38(2), 340-349.

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Antibody-Based Signaling Pathway Analysis

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All antibodies were purchased from commercial sources as follows: antibodies
against FGFR1 (monoclonal [rabbit], catalogue no. 9740, Antibody Registry
Identifier AB_11178519), ERK (monoclonal [mouse], 9107, AB_10695739),
phospho-p42/44 ERK1/2 (monoclonal [rabbit], 4370, AB_2315112), phospho-STAT5
(Tyr694; monoclonal [rabbit], 4322, AB_10544692), and STAT5 (monoclonal
[rabbit], 9358, AB_659905) were from Cell Signaling Technologies (Danvers, MA)
and used at 1:1000 dilution; FGFR3 (monoclonal [rabbit], AB133644, AB_2810262)
and FRS2 (polyclonal [rabbit], AB10425, AB_2247176) were from Abcam (Cambridge,
UK) and used at 1:2000; and phospho-FRS2 (Tyr436; polyclonal [rabbit], AF5126,
AB_2106234) was from R&D Systems (Minneapolis, MN) and used at 1 μg/mL. The
pFGFR2 enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D
Systems and ELISA was performed per manufacturer protocol.

Liu P.C., Koblish H., Wu L., Bowman K., Diamond S., DiMatteo D., Zhang Y., Hansbury M., Rupar M., Wen X., Collier P., Feldman P., Klabe R., Burke K.A., Soloviev M., Gardiner C., He X., Volgina A., Covington M., Ruggeri B., Wynn R., Burn T.C., Scherle P., Yeleswaram S., Yao W., Huber R, & Hollis G. (2020). INCB054828 (pemigatinib), a potent and selective inhibitor of fibroblast growth factor receptors 1, 2, and 3, displays activity against genetically defined tumor models. PLoS ONE, 15(4), e0231877.

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Protein Expression Analysis of Cellular Samples

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Cell lysates were subjected to polyacrylamide gel electrophoresis, and then blotted onto Immobilin-P polyvinylidene difluoride membranes (Millipore, Bedford, MA). Antibodies for cyclin D1 (catalog # 2978), CDK2 (catalog # 2546), phospho-Rb (catalog # 8516), Rb (catalog # 9309), PARP(catalog No # 9532), Caspase 3 (catalog # 9662), Bcl2 (catalog # 2876), BclXL (catalog # 2764), Bax (catalog No # 5023), CD44 (catalog # 3570), CD133 (catalog # 64326), phospho-STAT5 (catalog #s 4332, 9359), STAT5 (catalog # 25656), phospho-STAT3 (catalog # 9131), STAT3 (catalog # 4904), phospho-ERK (catalog # 4370) and ERK (catalog # 9102), ABCG2 (catalog #42078), DCLK1 (catalog # 62257) were purchased from Cell Signaling Technology (Danvers, MA, USA), for DCLK1 (catalog # ab37994), Oct-4 (catalog # ab189857) from Abcam Inc. (Cambridge, MA, USA), CDK4 (catalog # MA5–13498), CDK6 (catalog # MA5–13338) from Thermofisher (Waltham, MA, USA) and DCLK1 (catalog # SAB2420186) and Actin (catalog # A1978) from Sigma Aldrich and GAPDH (catalog # sc-365062) Santacruz Biotechnology Inc (Santa Cruz, CA, USA). Specific proteins were detected by chemiluminescence system.

Subramaniam D., Angulo P., Ponnurangam S., Dandawate P., Ramamoorthy P., Srinivasan P., Iwakuma T., Weir S.J., Chastain K, & Anant S. (2020). Suppressing STAT5 signaling affects osteosarcoma growth and stemness. Cell Death & Disease, 11(2), 149.

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Western Blot Analysis of Signaling Proteins

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Cells were washed with PBS and lysed in extraction buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF and protease inhibitor cocktails]. Cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore corporation, Billerica, MA, USA). Membranes were blocked with 5% skim milk in TBS-T [10 mM Tri-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20] and immunoblotted with antibodies against c-Fos (Santa Cruz Biotechnology, Dallas, TX, USA), NFATc1 (Santa Cruz Biotechnology), Phospho-STAT5 (Cell Signaling Technology), STAT5A (Cell Signaling Technology, Beverly, MA), IκB (Cell Signaling Technology), Flag-HRP (Sigma-Aldrich), Actin-HRP (Sigma-Aldrich), mouse-IgG-HRP (Abcam, Cambridge, UK) and rabbit-IgG-HRP (Abcam). Signals were detected with ECL solution (Millipore corporation) and analyzed using a LAS3000 luminescent image analyzer (GE Healthcare, Piscataway, NJ, USA).

Lee J., Seong S., Kim J.H., Kim K., Kim I., Jeong B.C., Nam K.I., Kim K.K., Hennighausen L, & Kim N. (2016). STAT5 is a key transcription factor for IL-3-mediated inhibition of RANKL-induced osteoclastogenesis. Scientific Reports, 6, 30977.

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Protein expression analysis by Western blot

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Western blots were generated by the semi-dry method. Proteins obtained from cell line lysates using SIGMAFast protease inhibitor cocktail (Sigma) were transferred onto nitrocellulose membranes (Bio-Rad, München, Germany) and blocked with 5% dry milk powder dissolved in phosphate-buffered-saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), AUTS2 (Origene), SMAD1 (Santa Cruz Biotechnology, Heidelberg, Germany), STAT5 (Santa Cruz Biotechnology), phospho-STAT5 (Cell Signaling Technology, Danvers, MA, USA). For loading control the blots were reversibly stained with Poinceau (Sigma) and then detection of alpha-Tubulin (TUBA) or SMAD1 was performed. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL (Perkin Elmer, Waltham, MA, USA). Documentation was performed using the digital system ChemoStar Imager (INTAS, Göttingen, Germany).

Nagel S., Pommerenke C., Meyer C., Kaufmann M., Drexler H.G, & MacLeod R.A. (2016). Deregulation of polycomb repressor complex 1 modifier AUTS2 in T-cell leukemia. Oncotarget, 7(29), 45398-45413.

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Comprehensive Protein Analysis in Tissues

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Immunohistochemistry, immunofluorescence, and immunoblot analyses were performed using monoclonal antibodies against FGF21, CLCA3, cleaved caspase 3, BrdU, β-Klotho (Abcam, Cambridge, MA, USA), Lysozyme (DAKO, Carpinteria, CA, USA), E-Cadherin, phospho-Stat3, Stat3, phospho-Stat5, Stat5, phospho-Protein kinase B (Akt), Akt, Socs2, Socs3, Janus-activated kinase (Jak) 1, Bax, β-actin (Cell Signaling Technologies, Beverly, MA, USA), and β-Klotho (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Liu L., Li F., Shao T., Zhang L., Lee J., Dryden G., McClain C.J., Zhao C, & Feng W. (2023). FGF21 Depletion Attenuates Colitis through Intestinal Epithelial IL-22-STAT3 Activation in Mice. Nutrients, 15(9), 2086.

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