Anti total stat3

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Anti-total STAT3 is a laboratory reagent that can be used to detect and quantify the total expression levels of the STAT3 protein. STAT3 is a transcription factor that plays a key role in cellular signaling pathways. This antibody can be used in various immunoassay techniques, such as Western blotting, to measure the overall abundance of STAT3 in biological samples.

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Close spelling variants of this product

STAT3 (1075 citations) Anti-STAT3 (536 citations) Total STAT3 (133 citations)

12 protocols using anti total stat3

Western Blot Analysis of Signaling Proteins

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Both liver tissues and cell lysates were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis before transferring to nitrocellulose membranes. After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: anti-phospho-STAT3, anti-total STAT3, anti-IκBα, anti-PKCδ, and anti-PTEN obtained from Cell Signaling Technology (Danvers, MA); anti-GSK3β from Abcam (Cambridge, UK); and anti-p38, anti-pERK, and anti-GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA). Membranes were washed and incubated with peroxidase-conjugated secondary antibodies (Amersham Bioscience, Amersham, UK).

Tian Y., Zhang M., Fan M., Xu H., Wu S., Zou S., Wang Y., Tang D., Zhang C., Han W., Yu H., Fu X, & Huang W. (2022). A miRNA-mediated attenuation of hepatocarcinogenesis in both hepatocytes and Kupffer cells. Molecular Therapy. Nucleic Acids, 30, 1-12.

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Quantitative Western Blot Analysis

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Whole retina lysates were prepared as in (Yi et al., 2012b (link)). Briefly, freshly isolated retinas were lysed in buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% NP40, 0.05% SDS) containing a proteinase and phosphatase inhibitor cocktail on ice. Twenty micrograms of protein lysate was electrophoresed using 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gels and proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were probed with anti-phosphorylated Stat3 (1:200 dilution, Cell Signaling, Danvers, MA), anti-total Stat3 (1:200 dilution, Cell Signaling), and anti-β-actin (1:8000 dilution, Sigma, St. Louis, MO), followed by the appropriate secondary antibody that was conjugated to HRP. Signal detection was using the LumiGLO Peroxidase Chemiluminescent Substrate Kit (Kirkegaard & Perry Laboratories, Gaithersburg MD) and imaged with a Fuji Film Luminescent Image Analyzer. The ImageJ software (Abramoff et al, 2004) was used for quantification.

Patel A.K., Park K.K, & Hackam A.S. (2016). Wnt signaling promotes axonal regeneration following optic nerve injury in the mouse. Neuroscience, 343, 372-383.

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Molecular Mechanisms of Honokiol in Cancer

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Honokiol (≥ 98% of purity) was purchased from LKT laboratories (Minneapolis, MN). Anti-phospho-EGFR (Y1068), anti-total EGFR, anti-phospho-Akt (S473), anti-total Akt, anti-phospho-ERK (T202/Y204), anti-total ERK, anti-phospho-STAT3 (Y705), anti-total STAT3, anti-cyclin D1, anti-CDK2, anti-CDK4, anti- phospho-Rb (S807/811), anti-p27, anti-caspase 3, anti-phospho-GSK3α/β (S21/9), anti-IκBα, anti-bax, anti-phospho-Bad, anti-β-actin, anti-caspase 8, anti-caspase 9 and goat anti-rabbit IgG secondary antibody were from Cell Signaling Technology (Beverly, MA). Anti-poly (ADP-ribose) polymerase (PARP) and anti-p21 were obtained from Santa Cruz Biotechnology. Erlotinib (99% of purity) was purchased from LC Laboratories (Woburn, MA). NNK (99% of purity) was synthesized as described elsewhere [60 (link)].

Song J.M., Anandharaj A., Upadhyaya P., Kirtane A.R., Kim J.H., Hong K.H., Panyam J, & Kassie F. (2016). Honokiol suppresses lung tumorigenesis by targeting EGFR and its downstream effectors. Oncotarget, 7(36), 57752-57769.

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Lung Cancer Tumorigenesis Protocols

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NTCU and LPS were purchased from Toronto Research Chemicals (Toronto, Canada) and
Sigma (St. Louis, MO), respectively. BioResponse diindolylmethane (DIM) was kindly
provided by Dr. Michael Zeligs (BioResponse, LLC). Anti-phospho-STAT3, anti-total STAT3,
anti-phospho-Akt, anti-total Akt, anti-phospho-extracellular signal-regulated kinase
(ERK), anti-total ERK, anti-phospho-p38, total p-38, anti-Mcl-1, anti-p53, anti-COX2,
anti-phospho IκBα, anti-total IκBα, anti-Bax, anti-p-21,
anti-PARP, anti-β-actin and goat anti-rabbit IgG secondary antibody were from Cell
Signaling Technology (Beverly, MA). Mouse diets (AIN-93G and AIN-93M) were purchased from
Harlan Teklad (Madison, WI). These diets are standard diets for lung tumorigenesis studies
in A/J mice. The AIN-93G diet, high in protein and fat, was used to support rapid growth
of the mice until eight weeks of age. AIN-93G diet was then replaced by AIN-93M diet, a
low-protein and low-fat diet, which is recommended for adult maintenance.

Song J.M., Qian X., Teferi F., Pan J., Wang Y, & Kassie F. (2014). Dietary diindolylmethane suppresses inflammation-driven lung squamous cell carcinoma in mice. Cancer prevention research (Philadelphia, Pa.), 8(1), 77-85.

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Immunoblotting Analysis of STAT3 and β-Catenin

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BMDM lysates were run in 12% SDS-polyacrylamide gels under denaturing conditions and blots were stained with anti-pY-STAT3 (9131;Cell Signaling Technology), anti-total-STAT3 (8768; Cell Signaling Technology), β-catenin (sc-7963; Santa Cruz), and anti-GAPDH antibodies (G8795; Sigma-Aldrich). Immunoreactive bands were visualized using the Odyssey imaging system (LI-COR Biosciences) by using horseradish peroxidase-conjugated secondary antibodies (ab205718; Abcam and 626520; Thermo Fisher Scientific) and chemiluminescence.

Weinstock A., Rahman K., Yaacov O., Nishi H., Menon P., Nikain C.A., Garabedian M.L., Pena S., Akbar N., Sansbury B.E., Heffron S.P., Liu J., Marecki G., Fernandez D., Brown E.J., Ruggles K.V., Ramsey S.A., Giannarelli C., Spite M., Choudhury R.P., Loke P, & Fisher E.A. (2021). Wnt signaling enhances macrophage responses to IL-4 and promotes resolution of atherosclerosis. eLife, 10, e67932.

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Western Blot Protein Detection Protocol

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Cells were rinsed in ice-cold PBS then lyzed in RIPA buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (ROCHE), 5 mM sodium fluoride, 2 mM sodium orthovanadate and 0.2 mM PMSF incubating on ice for 15 min. Lysates were cleared by centrifugation at 20,000 g for 15 min at 4°C then protein concentrations determined using Coomassie Protein Assay Kit (Thermo Scientific, UK). For each sample, 30 μg of total protein were separated on 4%–12% Bis-Tris polyacrylamide gels (NuPAGE, Invitroge) in MES SDS running buffer then blotted onto Protran 0.2 mM Nitrocellulose (GE Healthcare, UK). Membranes were probed with 1:1000 dilution of the appropriate primary antibody anti-total-STAT3 (Cell Signaling, #9139S), anti-total-RPB1 (Cell Signaling, #14958), anti-pSer2-RPB1 (CellSignaling, #13499), anti-pSer5-RPB1 (CellSignaling, #13523) and anti-GAPDH (Cell Signaling, #2118S). 1:5000 dilution of donkey anti-rabbit-HRP (Stratech, 711-035-152-JIR) or donkey anti-mouse-HRP (Stratech, 715-035-150-JIR) as the secondary antibody. Immobilon Western Chemiluminescent HRP substrate (Millipore, UK) was used for visualization.

Martinez-Fabregas J., Wang L., Pohler E., Cozzani A., Wilmes S., Kazemian M., Mitra S, & Moraga I. (2020). CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci. Cell Reports, 33(12), 108545.

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Immunoblotting for Protein Analysis

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Cell lysates were separated by appropriate sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods and transferred to nitrocellulose membranes. After blocking in 5% nonfat milk, membranes were incubated with the following primary antibodies: anti-phospho-STAT3, anti-total STAT3, and anti- IκBα, obtained from Cell Signaling Technology (Danvers, MA, USA) and anti-c-myc, anti-PCNA, and anti-GAPDH, purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were washed and reacted with peroxidase-conjugated secondary antibodies (Amersham Biosciences, UK).

Zhang W., Fu X., Xie J., Pan H., Han W, & Huang W. (2021). miR-26a attenuates colitis and colitis-associated cancer by targeting the multiple intestinal inflammatory pathways. Molecular Therapy. Nucleic Acids, 24, 264-273.

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Signaling Pathway Activation Assay

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Recombinant human IL-6 was purchased from R & D systems (Minneapolis, MN, USA). Anti-phospho STAT3 (Tyr⁷⁰⁵) antibody was purchased from Calbiochem (Darmstadt, Germany) and anti-total STAT3, anti-phospho JAK2 (Tyr^1007/1008), anti-phospho ERK (Tyr^202/204) were from Cell Signaling Technology (Boston, MA, USA). All reagents including genistein were obtained from Sigma-Aldrich Ltd (St Louis, MO, USA).

Hwang J.T., Kim Y., Jang H.J., Oh H.M., Lim C.H., Lee S.W, & Rho M.C. (2016). Study of the UV Light Conversion of Feruloyl Amides from Portulaca oleracea and Their Inhibitory Effect on IL-6-Induced STAT3 Activation. Molecules, 21(7), 865.

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Quantitative Analysis of MAOA and STAT3 Proteins

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Protein expression of MAOA and STAT3 was examined by Western blotting as previously described^{43 (link)}. Cells were lysed in ice-cold NP40 lysis buffer [150 mM NaCl, 1% IGEPAL CA-630, 50 mM Tris-HCl (pH 8.0)] containing protease inhibitors (cocktail set III; Calbiochem, Merck Millipore) and phosphatase inhibitors (Halt phosphatase inhibitor cocktail; Thermo Scientific, USA). Samples containing equal amounts of protein were separated under reducing conditions using SDS-PAGE and the proteins transferred to polyvinylidene difluoride membranes (Millipore, UK). The primary antibodies used in this study were anti-MAOA (1:1000; Abcam, UK), anti-phospho-STAT3 (Tyr705; 1:1000; Cell Signaling), anti-total STAT3 (1:1000; Cell Signaling) and anti-β-actin (1:5000; Sigma-Aldrich, USA). Bound antibodies were detected with peroxidase conjugated secondary antibodies and Enhanced Chemiluminescence reagents (Advansta, USA).

Lee H.M., Sia A.P., Li L., Sathasivam H.P., Chan M.S., Rajadurai P., Tsang C.M., Tsao S.W., Murray P.G., Tao Q., Paterson I.C, & Yap L.F. (2020). Monoamine oxidase A is down-regulated in EBV-associated nasopharyngeal carcinoma. Scientific Reports, 10, 6115.

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Antibody-based immunoblotting protocol

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Sample preparation and immunoblotting were carried out as previously described^{25 (link)}. The following antibodies from Cell Signalling Technologies (MA, US) were used: anti-phospho-Stat3 (Tyr705: 9131), anti-total-Stat3 (9132), anti-phospho-Akt (Ser473: 9271), anti-total-Akt (9272), anti-phospho-Gsk3β (9331), anti-GSK3β (9315), anti-Akt1 (2967), anti-Akt2 (2964), anti-Akt3 (4059) and anti-Lamin A/C (2032). The following antibodies from Abcam (Cambridge, UK) were used: anti-cathepsin B (ab33538), anti-Lamp2 (ab13524), anti-Tubulin (ab6160) and anti-β-actin (ab8227). The following antibodies from Santa Cruz Biotechnology (CA, US) were used: anti-C/ebpα (sc-61), anti-histone H3 (sc8654), anti-Bax (sc-7480), anti-Bcl-2 (sc7382) and anti-Bcl-xL (sc-634). Other commercial antibodies used were: anti-cathepsin L (MAB9521), anti-cathepsin L (AF1515, used for the immunodetection of cathepsin L in the cathepsin L knockout and control glands) and anti-Bid (MB860) from R&D Systems (MN, US), anti-pan-p85 (Millipore, 06-496, also detects p50α/p55α subunits), anti-cytochrome c (65981A) and anti-E-cadherin (610182) from BD biosciences. All antibodies were used at a standard dilution of 1:1,000. Secondary horseradish peroxidase (HRP)-conjugated antibodies were purchased from Dako (Glostrup, Denmark).

Pensa S., Neoh K., Resemann H.K., Kreuzaler P.A., Abell K., Clarke N.J., Reinheckel T., Kahn C.R, & Watson C.J. (2014). The PI3K regulatory subunits p55α and p50α regulate cell death in vivo. Cell Death and Differentiation, 21(9), 1442-1450.

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