Stat5

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

STAT5 is a laboratory product from Cell Signaling Technology. It is a protein that plays a core role in cellular signaling pathways.

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129 protocols using stat5

Western Blot Analysis of Phosphorylated STAT5

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T cells were plated overnight in RPMI + 10% FBS and glutamax without additional cytokine. T cells were lysed in RIPA lysis buffer with protease (cOmplete) and phosphatase (PhosSTOP) inhibitor cocktails (MilliporeSigma) on ice. Electrophoresis was conducted using Novex WedgeWell 10% Bis-Tris Mini Gels (Thermo Fisher Scientific) and protein transferred to polyvinylidene difluoride (PVDF) membrane. Western blot analysis was performed with the following antibodies: rabbit anti-human p-STAT5 (done D47E7, Cell Signaling Technology), STAT5 (done D2605, Cell Signaling Technology), and GAPDH (done 6C5, Invitrogen).

Krawczyk E., Zolov S.N., Huang K, & Bonifant C.L. (2019). T-cell Activity against AML Improved by Dual-Targeted T Cells Stimulated through T-cell and IL7 Receptors. Cancer immunology research, 7(4), 683-692.

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Immunoblot Analysis of STAT Proteins

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Immunoblots were prepared as described^{41 (link)} using Abs for STAT3 (79D7, #4904), pSTAT3 (Y705, #9145), STAT5 (#9363), pSTAT5 (Y694, 9351), p STAT1 (Y701, #9171), β-actin (#4967) (Cell Signaling Technology, Danvers, MA, USA), or STAT1 (#610185) (BD Transduction Labs, San Jose, CA, USA). Following incubation with horseradish peroxidase-conjugated secondary Ab, immune complexes were detected via SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

Ware M.B., Zaidi M.Y., Yang J., Turgeon M.K., Krasinskas A., Mace T.A., Keenan K., Farren M.R., Ruggieri A.N., Li Y., Zhang C., Chen Z., Young G.S., Elnaggar O., Che Z., Maithel S.K., Bekaii-Saab T., El-Rayes B, & Lesinski G.B. (2020). Suppressive myeloid cells are expanded by biliary tract cancer-derived cytokines in vitro and associate with aggressive disease. British Journal of Cancer, 123(9), 1377-1386.

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Western Blot Analysis of STAT3/STAT5 Signaling

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Western blot was performed as described previously with the following modifications^{30 (link)}. RIPA buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich) was used to prepare the whole-cell lysate. Twenty micrograms protein/sample was used for western blot. BSA (Sigma-Aldrich) was used instead of non-fat dry milk during blocking. The primary antibodies used for western blotting are as follows: STAT3 (Cell Signaling Inc., Danvers, MA), STAT5 (3H7) Rabbit mAb #9358 (Cell Signaling), phospho-STAT5 D47E7 Rabbit mAb # 4322 (Cell Signaling), phospho-STAT3 (Cell Signaling) and α-Tubulin (Sigma-Aldrich). The working dilution of α-Tubulin is 1:50,000. The working dilution for all other antibodies is 1:1,000. Uncropped representative WB images are shown in Supplementary Fig. 5.

Küçük C., Jiang B., Hu X., Zhang W., Chan J.K., Xiao W., Lack N., Alkan C., Williams J.C., Avery K.N., Kavak P., Scuto A., Sen E., Gaulard P., Staudt L., Iqbal J., Zhang W., Cornish A., Gong Q., Yang Q., Sun H., d’Amore F., Leppä S., Liu W., Fu K., de Leval L., McKeithan T, & Chan W.C. (2015). Activating mutations of STAT5B and STAT3 in lymphomas derived from γδ-T or NK cells. Nature communications, 6, 6025.

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Temporal Signaling Dynamics in C2C12 Myotubes

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Four-day differentiated C2C12 myotubes were treated at the time points indicated, washed twice in 1X PBS (Thermo Fisher Scientific), and lysed in radioimmunoprecipitation assay (RIPA) buffer (MilliporeSigma) on ice for 30 minutes. Cells were then scraped into 1.7mL Eppendorf tubes, centrifuged at 9000× g for 10 minat 4 °C, and supernatant collected. Protein lysates were quantified by BCA assay (Thermo Fisher Scientific) according to the manufacturer’s instructions. Equal amounts of protein were separated by SDS-PAGE (4%–15% TGX stain-free gel Bio-Rad, Hercules, CA, USA), and proteins transferred using the trans-blot turbo transfer system (Bio-Rad). The nitrocellulose membranes were probed with the following antibodies from Cell Signaling (Danvers, MA, USA), according to established Western blot protocols: p-STAT3 (#9145), STAT3 (#9139), p-STAT5 (#9351), STAT5 (#94205), p-SMAD2 (#3104), p-SMAD3 (#9520), SMAD2/3 (#8685), p-ERK1/2 (#4370), ERK1/2 (#4695), IκBα (#4814), p-p38 (#9216), p38 (#9212), and GAPDH (#2118). The secondary signal was quantified by fluorescence using a LI-COR Odyssey^® imager (LI-COR Biosciences, Lincoln, NE, USA), and the signal was normalized to a GAPDH loading control.

Callaway C.S., Delitto A.E., D’Lugos A.C., Patel R., Nosacka R.L., Delitto D., Deyhle M.R., Trevino J.G., Judge S.M, & Judge A.R. (2019). IL-8 Released from Human Pancreatic Cancer and Tumor-Associated Stromal Cells Signals through a CXCR2-ERK1/2 Axis to Induce Muscle Atrophy. Cancers, 11(12), 1863.

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Whole Cell Protein Extraction and Western Blotting

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For whole cell protein extracts, cells were collected and lysed with RIPA buffer (Boston Bioproducts, Ashland, MA). Protein was quantitated using the BCA assay (Pierce, Rockford, IL). Lysates were electrophoretically separated using 12% Tris HCl gels (Biorad, Hercules, CA) and transferred to nitrocellulose membranes. Western blotting was performed using antibodies specific for the following proteins: p STAT5, STAT5, pAkt, Akt, p56lck, phosphor-p56lck, p Erk1/2, Erk1/2, p FoxO1/FoxO3a, FoxO1, GATA-1 (all from Cell Signaling Technologies, Danvers, MA), NFATc1, goat anti-rabbit IgG-HRP, and goat anti-mouse IgG-HRP. Average densitometric ratio was calculated for each blot by normalizing to an actin loading control using Image J software.

Grishkan I.V., Tosi D.M., Bowman M.D., Harary M., Calabresi P.A, & Gocke A.R. (2015). Antigenic stimulation of Kv1.3-deficient helper T cells gives rise to a population of Foxp3-independent T cells with suppressive properties. Journal of immunology (Baltimore, Md. : 1950), 195(4), 1399-1407.

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Quantitative Western Blot Analysis of Signaling Pathways

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Western blot was performed as previously described (4 (link)). Antibodies against FAK (cat#13009s), p-STAT5
(cat#9351s), STAT5 (cat#9363s), p-AKT (cat#4060s), AKT (cat#2920s), BCL-XL
(cat#2764s), p-ERK (cat#4370s), 4EBP1 (cat#9644), and p-4EBP1 (cat#2855) were
purchased from Cell Signaling Technology (Danvers, MA). Antibodies against MCL-1
(cat#sc-819) and BCL-2 (cat#M0887) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA) and DAKO (Carpentaria, CA), respectively. β-actin or
α-tubulin was used as a loading control. Signals were detected using the
Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) and quantified
using Odyssey software (version 3.0, LI-COR Biosciences).

Wang X., Mak P.Y., Mu H., Tao W., Rao A., Visweswaran R., Ruvolo V., Pachter J.A., Weaver D.T., Andreeff M., Xu B, & Carter B.Z. (2020). Combinatorial inhibition of focal adhesion kinase and BCL-2 enhances anti-leukemia activity of venetoclax in acute myeloid leukemia. Molecular cancer therapeutics, 19(8), 1636-1648.

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Western Blot Analysis of Protein Targets

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Western blots were performed as previously described [21] (link), using the following antibodies detecting: Akt (cat.# 4691, Cell signalling, dilution 1:1000), Dnmt1 (cat.# ab188453, Abcam; dilution 1:500), Dnmt3a (cat.# SC-20703, Santa Cruz; dilution 1:1000), Dnmt3b (cat.# PA1–884, Thermo Fisher; dilution 1:1000), Stat3 (cat.# SC-8019, Santa Cruz; dilution 1:500), P-Stat3 (cat.# 9145, Cell signalling, dilution 1:1000), Stat5 (cat.# 25656, Cell signalling, dilution 1:1000), P-Stat5 (cat.# 9359, Cell signalling, dilution 1:1000), mouse c-Met (cat.# SC-8057; Santa Cruz; dilution 1:500), c-Myc (cat.# SC-40, Santa Cruz; dilution 1:1000), Hsc-70 (cat.# SC-7298, Santa Cruz; dilution 1:10000), Lin28b (cat.# SC-293120, Santa Cruz; dilution 1:500), h-Ras (cat.# SC-520, Santa Cruz, dilution 1:1000) and β-actin (cat.# SC-130657, Santa Cruz; dilution 1:1000).

Lopusna K., Nowialis P., Opavska J., Abraham A., Riva A, & Opavsky R. (2021). Dnmt3b catalytic activity is critical for its tumour suppressor function in lymphomagenesis and is associated with c-Met oncogenic signalling. EBioMedicine, 63, 103191.

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Protein Expression Analysis Protocol

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Abs against phospho-ATM (Ser1981), ATM, phospho-Chk1 (Ser317), Chk1, phospho-Chk2 (Thr68), Chk2, phospho-p53 (Ser15), PTEN, phospho-CDK2 (Thr160), CDK2, Cyclin E, Cyclin B1, CDC2, phospho-CDC2 (Tyr15), cleaved PARP (Asp214), phospho-STAT3 (Ser727), STAT3, STAT5, caspase-3 and active caspase-3 (Asp175) were all obtained from Cell Signaling (Danvers, MA, USA). Abs recognizing p53 (EMD Millipore, Darmstadt, Germany), p21 (Santa Cruz, Dallas, Texas) and phospho-STAT5 (Ser726/731) (Sabbiotech, College Park, MD, USA) were obtained as indicated.

Chen L.M., Liu P.Y., Chen Y.A., Tseng H.Y., Shen P.C., Hwang P.A, & Hsu H.L. (2017). Oligo-Fucoidan prevents IL-6 and CCL2 production and cooperates with p53 to suppress ATM signaling and tumor progression. Scientific Reports, 7, 11864.

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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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Protein Isolation and Immunoblotting from Tissues

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Protein from snap frozen tissues was isolated as before[49 (link)]. Protein from cell pellets was isolated by pipetting in NP-40 cell lysis buffer (Life Technologies) containing 1% PMSF, incubating on ice for 30 minutes, and removing cleared lysate. Antibodies were purchased from Cell Signaling Technology (p-KIT, Y719, #3391; KIT, D13A2, #3074; p-AKT, Ser473, #4060; AKT, 11E7, #4685; p-S6, S235/236, #2211; S6, 5G10, #2217; p-ERK, T202/Y204, #4370; ERK1/2, #4695; p-STAT5, Tyr694, #9359; STAT5, D2O6Y, #94205; p-STAT3, Y705, #9145; STAT3, 79D7, #4904; p-Tyrosine, p-Tyr-1000, #8954; GAPDH, #5174).

Zhang J.Q., Bosbach B., Loo J.K., Vitiello G.A., Zeng S., Seifert A.M., Medina B.D., Param N.J., Maltbaek J.H., Rossi F., Antonescu C.R., Besmer P, & DeMatteo R.P. (2020). The V654A second-site KIT mutation increases tumor oncogenesis and STAT activation in a mouse model of gastrointestinal stromal tumor. Oncogene, 39(49), 7153-7165.

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