Stat3

Manufactured by Thermo Fisher Scientific

Sourced in United States

STAT3 is a lab equipment product offered by Thermo Fisher Scientific. It is a key component in cellular signaling pathways and plays a role in various biological processes. The core function of STAT3 is to facilitate signal transduction and regulate gene expression.

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STAT3 siRNA (17 citations) Anti-STAT3 (12 citations) Anti-p-STAT3 (6 citations) P-STAT3 (Y705) (4 citations) GIPZ lentiviral LIFR- and STAT3-targeting human shRNAs (2 citations) P-STAT3–S727–PE (2 citations) Phospho-STAT3 ELISA (2 citations) STAT3(pY705) (2 citations) ShRNA targeting Stat3 (2 citations) PE-conjugated anti-p-STAT3 (1 citations)

25 protocols using stat3

SNP Genotyping of Inflammatory Genes

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Approximately 2 mL of peripheral blood from each patient was collected into ethylenediaminetetraacetic acid (EDTA) tubes. Genomic DNA was extracted from leukocytes using the salting-out method [15 (link)] and stored at −80 °C until further use. The Multiplex SNaPshot method [16 (link)] is a primer extension-based method for genotyping known SNP positions through the manuscript-automated DNA analyzer invented by Applied Biosystems, which was used for genotyping 25 tag SNPs in NLRP3, ELP2, STAT3, CASP1, NFKBIA, NFKB1, CARD8, and CD14 (Applied Biosystems, Foster City, CA, USA). Table S1 lists the forward (F), reverse (R), and extension primers used in the PCR and extension procedures [17 (link)]. The detailed protocols have been previously described [14 (link)].

Qu Y., Li J., Zhang W., Xia C., Ou S., Yang Y., Jiang N., Ma Y., Qi Y, & Xu C. (2023). Posttraumatic Osteomyelitis Risks Associated with NLRP3 Gene Polymorphisms in the Chinese Population. Journal of Personalized Medicine, 13(2), 253.

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Transient Transfection and RNAi Knockdown

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The transient transfection of expression constructs was carried out 24 h after seeding using Transfast (Promega, Mannheim, Germany) and Lipofectamine™ 2000 (Invitrogen, Carlsbad, USA) as transfection reagents following manufacturer’s instructions. Small interfering RNA (siRNA) was transfected using the siLentFect™ Lipid Reagent (Bio-Rad, München, Germany) according to the manufacturer’s protocol. The RNAi oligonucleotide sequences were as follows: NFATc2 5’-ccauuaaacaggagcagaatt-3’; STAT3: 5’-gcacaaucuacgaagaauctt-3’ (Applied Biosystems, Austin, USA); GSK-3β: siGENOME ON-TARGET plus SMARTpool duplex (Thermo Scientific, Dreieich, Germany). The Silencer Negative Control from Ambion (Applied Biosystems) was used as a negative control.

Baumgart S., Chen N.M., Zhang J.S., Billadeau D.D., Gaisina I., Kozikowski A.P., Singh S., Fink D., Ströbel P., Klindt C., Zhang L., Bamlet W.R., Koenig A., Hessmann E., Gress T., Ellenrieder V, & Neesse A. (2016). GSK-3β governs inflammation-induced NFATc2 signaling hubs to promote pancreatic cancer progression. Molecular cancer therapeutics, 15(3), 491-502.

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Systematic Gene Expression Analysis in Muscle Tissue

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RNA was extracted from TA and diaphragm muscles using TRIzol as previously described [16 (link)]. Isolated total RNA was subsequently purified using an RNeasy Mini kit (Qiagen, Valencia, CA), according to manufacturer’s instructions. The resulting quantity and purity of total RNA was tested through absorbance spectrophotometry at 230, 260 and 280 nm, and the quality of RNA was tested on a 1% denaturing agarose gel. Synthesis of cDNA and qRT-PCR analyses from RNA isolated from the TA and diaphragm were performed as described previously [28 (link)] using a 7300 real-time PCR system and the following primers from Applied Biosystems (Austin, TX): Fbxo30 (NM_027968.3), Fbxo31 (NM_133765.4), Bach2 (NM_001109661.1), Socs3 (NM_007707.3), Ubr2 (NM_146078.3), Psma2 (NM_008944.2), Ubqln1 (NM_026842.4), Fos (NM_010234.2), Cebpb (NM_009883.3), Stat3 (NM_011486.4), Col6a2 (NM_146007.2), Myoz3 (NM_133363.3), atrogin-1/MAFbx/Fbxo32 (NM_026346.2), MuRF1/Trim63 (NM_001039048.2), Bcl3 (NM_033601.3), and Maff (NM_010755.3).

Judge S.M., Wu C.L., Beharry A.W., Roberts B.M., Ferreira L.F., Kandarian S.C, & Judge A.R. (2014). Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia. BMC Cancer, 14, 997.

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Validating Gene Silencing in Macrophages

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To confirm successful gene silencing, total RNA was extracted from macrophages using the RNeasy Mini Kit (Qiagen, Venlo, Limburg, Netherlands). 200 ng RNA was then reverse transcribed to generate cDNA templates as described previously (Piccinini and Midwood, 2012 (link)). Expression of mRNA was determined using TaqMan® Universal PCR Master Mix and primer/probe sets for: STAT-3 (Hs00374280_m1), SRC (Hs01082246_m1), MAPK14 (Hs00176247_m1), MAPK12 (Hs00268060_m1), SYK (Hs00895377_m1), HCK (Hs00176654_m1), JAK1 (Hs01026983_m1), JAK3 (Hs00169663_m1), PI3KCD (Hs00192399_m1), PI3KCG (Hs00277090_m1) (Applied Biosystems). Reactions were performed in a Rotor-Gene 6000 instrument (Corbett Life Science, Qiagen, Venlo, Limburg, Netherlands) and relative gene expression was determined by ΔΔC_t algorithm (Ruhmann et al., 2012 (link)).

To W.S., Aungier S.R., Cartwright A.J., Ito K, & Midwood K.S. (2015). Potent anti-inflammatory effects of the narrow spectrum kinase inhibitor RV1088 on rheumatoid arthritis synovial membrane cells. British Journal of Pharmacology, 172(15), 3805-3816.

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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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Phosphorylation-mediated signaling analysis

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Primary antibodies: Phospho-STAT3 (Tyr705) (Thermo Fisher, USA; Cat# 44380G), p-NFκB p65 (S536) (Thermo Fisher, USA; Cat# MA515160), STAT3 (Thermo Fisher, USA; Cat# 10253-2-AP), beta Crystallin A3 (Abcam, USA; Cat# ab151722), IL-6 (Biorbyt, USA; Cat# orb6210), IL-1α (Biorbyt, USA; Cat# orb184287) and mNeonGreen (Chromotek, USA; Cat# 3216-100), Secondary antibodies: HRP anti-Rabbit IgG (KPL, USA; Cat# 074-1506), HRP anti-tagged anti-Mouse IgG (KPL, USA; Cat# 5220-0341), HRP anti-tagged Goat IgG (KPL, USA; Cat# 14-13-06). GIPZ Cryba1 shRNA Viral Particle Starter Kit (Dharmacon, USA; Cat# VGH5526-EG12957), AAV2-GFP-U6-shRNA (Vector Biolabs, USA; Cat# 7041). PTP1B (Ad-CMV-mNeonGreen-mPtpn1; Cat# AAV-269791) and Cryba1 (Ad-CMV-RFP-mCryba1; Cat# 2001) overexpression vectors were purchased from Vector Biolabs, USA. ELISA kits: Human crystallin, beta A1 (CRYBA1) (Cat# MBS7252187) and protein tyrosine phosphatase 1B (PTP1B) (Cat# MBS761801) ELISA kits were purchased from Mybiosource, USA. Proteins: CRYBA1 (NM_005208) purified human protein (OriGene Technologies, Cat# TP321965), Recombinant human PTP1B protein (Abcam, Cat# ab51277).

Ghosh S., Liu H., Yazdankhah M., Stepicheva N., Shang P., Vaidya T., Hose S., Gupta U., Calderon M.J., Hu M.W., Nair A.P., Weiss J., Fitting C.S., Bhutto I.A., Gadde S.G., Naik N.K., Jaydev C., Lutty G.A., Handa J.T., Jayagopal A., Qian J., Sahel J.A., Rajasundaram D., Sergeev Y., Zigler JS J.r., Sethu S., Watkins S., Ghosh A, & Sinha D. (2021). βA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity. Communications Biology, 4, 248.

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Immunohistochemical Analysis of Skin Samples

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Immunohistochemical (IHC) studies were performed on 2‐μm‐thick paraffin‐embedded skin sections, which were rehydrated in xylene and increasing dilutions of ethanol. Antigen retrieval was performed using a TRIS‐EDTA buffer solution (pH 9.0). The following antibodies were used: CD3 (#RM9107‐S0, Thermo Fisher Scientific, USA), STAT3 (9D8; #MA1‐13042, Thermo Fisher Scientific), STAT5A (E289; #ab32043, Abcam, UK), STA5B (#ab235934, Abcam), and pY‐STAT5 (Tyr694/699; #9359, Cell Signaling Technologies/CST, USA). The stained slides were imaged/scanned by the Panoramic Digital Slide Scanner (3DHistech. Ltd, Hungary) and Aperio Digital Pathology Slide Scanners (Leica Biosystems, Germany). IHC images were analyzed with the CaseViewer software (3DHistech. Ltd) and QuPath (Bankhead et al, 2017 (link)).

Sorger H., Dey S., Vieyra‐Garcia P.A., Pölöske D., Teufelberger A.R., de Araujo E.D., Sedighi A., Graf R., Spiegl B., Lazzeri I., Braun T., Garces de los Fayos Alonso I., Schlederer M., Timelthaler G., Kodajova P., Pirker C., Surbek M., Machtinger M., Graier T., Perchthaler I., Pan Y., Fink‐Puches R., Cerroni L., Ober J., Otte M., Albrecht J.D., Tin G., Abdeldayem A., Manaswiyoungkul P., Olaoye O.O., Metzelder M.L., Orlova A., Berger W., Wobser M., Nicolay J.P., André F., Nguyen V.A., Neubauer H.A., Fleck R., Merkel O., Herling M., Heitzer E., Gunning P.T., Kenner L., Moriggl R, & Wolf P. (2022). Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T‐cell lymphoma. EMBO Molecular Medicine, 14(12), e15200.

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Antibody Characterization for Immunoassays

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The following antibodies and dilutions were used: Actin (CST, 13E5, 1:1000 in 3% milk); Cluster of Differentiation 4 (CD4); Affymetrix eBioscience/Thermo Fisher; clone 4SM95; 14-97664; 1:100 (IHC); CD8a (Synaptic Systems, Göttingen, Germany) 361003; 1:500 (IHC); Gapdh (Calbiochem, #CB1001, Darmstadt, Germany) 1:20,000; Iba1 (Wako, 019-19741); 1:1000 (IHC); Ki67 (SP6, Thermo Fisher, MA5-14520); 1:100 (IHC); Stat3 (Santa Cruz Biotechnology (Heidelberg, Germany)), c-20; sc-482; 1:200 (WB); Stat3 (phospho Tyr705; CST Technologies (CST (Frankfurt am Main, Germany)); D3A7; 9145S; 1:1000 for Western Blot (WB) and 1:500 for Immunohistochemistry (IHC)); Stat3 (Thermo Fisher, 9D8, 1:5000 in 3% milk); Vinculin (Sigma Aldrich, hVIN-1, 1:2000 in 3% milk).

Linder B., Weirauch U., Ewe A., Uhmann A., Seifert V., Mittelbronn M., Harter P.N., Aigner A, & Kögel D. (2019). Therapeutic Targeting of Stat3 Using Lipopolyplex Nanoparticle-Formulated siRNA in a Syngeneic Orthotopic Mouse Glioma Model. Cancers, 11(3), 333.

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Quantitative Protein Analysis in Tumor Samples

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Total protein was extracted from thawed tumors using Cell Extraction Buffer (Invitrogen) containing 1 mM PMSF, 1X protease inhibitor cocktail, and 1X EDTA (Thermo Scientific) and quantified using pierce BCA protein assay (Thermo Scientific) as directed. Protein lysates [40 μg] were resolved by SDS-PAGE (Mini-PROTEAN TGX Precast Protein Gels, Bio-Rad) and transferred (Mini Trans-Blot® Cell, BioRad) to PVDF membranes as directed. PVDF membranes were incubated with rocking in Blocking Buffer [5% (w/v) nonfat dry milk in TBST Buffer] (r.t., 1 h), rinsed with TBST (r.t., 5 sec), incubated with primary antibodies (β-Actin [sc-47778] and Stat3 [sc-8019], Santa Cruz Biotechnology) [1:200 in TBST] (4°C, overnight), rinsed 3X with TBST (r.t., 5 min.), and incubated with secondary antibody (horseradish peroxidase-conjugated mouse IgGκ binding protein; m-IgGκ BP-HRP [sc-516102], Santa Cruz Biotechnology) [1:5000 in TBST (r.t., 1 h), and rinsed 3X with TBST as above. The blot was incubated with HRP substrate (Luminata Classico Western HRP, Sigma) as directed, visualized (MyECL imager, Thermo Scientific), and the average ratios of Stat3 / β-Actin protein band intensities ±SD (n=2 measurements) were determined by imaging densitometry (ImageJ software).

Ye Z., Abdelmoaty M.M., Ambardekar V.V., Curran S.M., Dyavar S.R., Arnold L.L., Cohen S.M., Kumar D., Alnouti Y., Coulter D.W., Singh R.K, & Vetro J.A. (2021). Preliminary Preclinical Study of Chol-DsiRNA Polyplexes Formed with PLL[30]-PEG[5K] for the RNAi-Based Therapy of Breast Cancer. Nanomedicine : nanotechnology, biology, and medicine, 33, 102363.

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Gene Expression Analysis of Inflamed Skin

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To analyze gene expression in skin sections, 10 cryosections (12 µm) were prepared and used for RNA isolation, reverse transcription, and real-time RT-PCR as previously described (23 (link)). In short, total RNA was isolated from comparable inflamed lesional skin of both groups according to the manufacturer’s protocol (innuPrep RNA Mini Kit, Analytic Jena AG). After reverse transcription, the cDNA was added to either qPCR Master MixPlus or qPCR Master Mix SYBR Green Plus (Thermo Fisher Scientific) and amplified using an SDS ABI7900 system (Applied Biosystems, Darmstadt, Germany). The number of cDNA copies was normalized using the 2^ΔCT method with housekeeping gene Gapdh.
Markers selected for analysis were Il17a (Mm00439618_m1), Itgam (Mm00434455_m1), Cxcl1 (Mm04207460_m1), Ifngr1 (Mm00599890_m1), Tnf (Mm00443258_m1), Il4ra (Mm01275139_m1), Csf2 (Mm00438328_m1), Stat3 (Mm01219775_m1), Il10 (Mm01288386_m1), Stat6 (Mm01160477_m1), and Gapdh (Mm99999915_g1) all from Thermo Fisher Scientific.

Gross N., Marketon J., Mousavi S., Kalies K., Ludwig R.J, & Bieber K. (2024). Inhibition of interferon gamma impairs induction of experimental epidermolysis bullosa acquisita. Frontiers in Immunology, 15, 1343299.

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