P stat3

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Canada, Germany

P-STAT3 is a laboratory equipment product designed for the detection and quantification of phosphorylated STAT3 (Tyr705) protein in various biological samples. It is a tool used in research applications to study the activation and signaling pathways of the STAT3 transcription factor.

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STAT3 (186 citations) Anti-p-STAT3 (24 citations) P-STAT3 (Tyr 705) (5 citations) Anti-p-STAT3 antibody (3 citations) P-STAT3 antibody (3 citations) P-STAT3 (S727) (3 citations) Anti-p-STAT3 (Tyr705) (3 citations) P-Stat3 (SC-8059) (2 citations) Anti-p-STAT3 (Ser 727, (2 citations) P-STAT3(B-7)/sc-8059 (2 citations)

107 protocols using p stat3

Western Blot and Immunohistochemistry Analysis of Signaling Pathways

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All sources of antibodies with name of antibodies, dilution ratio of antibodies, catalog numbers, and company in Western blot analysis study as follows: p-ERK1/2 (1:2000, #9101S, Cell Signaling Technology, Beverly, MA), ERK1/2 (1:2000, #9102S, Cell Signaling), p-p38 (1:1000, #9211S, Cell Signaling), p38 (1:1000, #9212S, Cell Signaling), p-JNK (1:500, #9251, Cell Signaling), JNK (1:1000, #9252S, Cell Signaling), JAK2 (1:1000, #3230, Cell Signaling), p-JAK2 (1:1000, #4406, Cell Signaling), STAT3 (1:1000, #12640, Cell Signaling), p-CREB (1:1000, #9191, Cell Signaling), PTEN (1:1000, #9552, Cell Signaling), Survivin (1:1000, #2808, Cell Signaling), Caspase-3 (1:1000, #9665, Cell Signaling), PARP (1:1000, #9542, Cell Signaling), Bcl-xL (1:1000, #sc-7195, Santa Cruz), β-actin (C4) (1:1000, #sc-47778, Santa Cruz), p-STAT3 (1:1000, #sc-8001, Santa Cruz).
For immmo(cyto)histochemistry study: Ki-67 (1:200, #sc-15402, Santa Cruz), PCNA (1:200, #sc-56, Santa Cruz), Survivin (1:200, #2808, Cell Signaling), Bcl-xL (1:200, #sc-7195, Santa Cruz), p-STAT3 (1:200, #sc-8001, Santa Cruz)

Park K.R., Yun H.M., Quang T.H., Oh H., Lee D.S., Auh Q.S, & Kim E.C. (2016). 4-Methoxydalbergione suppresses growth and induces apoptosis in human osteosarcoma cells in vitro and in vivo xenograft model through down-regulation of the JAK2/STAT3 pathway. Oncotarget, 7(6), 6960-6971.

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Spinal Cord Tissue Immunostaining Protocol

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Spinal cord tissue segments were cut longitudinally, fixed in 10% paraformaldehyde for 24 h, embedded, and sectioned at 5 μm thickness. Hematoxylin-eosin staining was used to observe the morphological changes. Immunohistochemical staining was performed by the streptavidin-biotin complex methods. The sections were stained by the streptavidin-biotin complex kit (Boter, Wuhan, China). According to the streptavidin-biotin complex kit manufacturer's instructions, we have chosen the following primary monoclonal antibodies: IL-17 (Santa Cruz Biotechnology, USA) at a working concentration of 5 μg/mL and p-STAT3 (Santa Cruz Biotechnology) at a working concentration of 5 μg/mL. To assess the expressions of IL-17 and p-STAT3, we measured 10 samples in one section chosen at random from each spinal cord. Digital images of IL-17 and p-STAT3 immunohistochemistry were obtained using a light microscope (Eclipse E800, Nikon, Japan). The Software Image-Pro Plus Version 5.0 (Media Cybernetics, Bethesda, MD, USA) was used to measure the integrated optical density (IOD).

Zong S., Zeng G., Fang Y., Peng J., Tao Y., Li K, & Zhao J. (2014). The Role of IL-17 Promotes Spinal Cord Neuroinflammation via Activation of the Transcription Factor STAT3 after Spinal Cord Injury in the Rat. Mediators of Inflammation, 2014, 786947.

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Immunoblotting and Flow Cytometry of Cell Signaling Proteins

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Mouse monoclonal anti-human NOS1 (nNOS) (MAB2416-SP, Novus Biologicals, Centennial, CO), STAT3, p-STAT3, Lamin A/C (sc-8019; sc-8059; sc-398927; Santa Cruz Biotechnology, Dallas, TX), rabbit monoclonal STAT1 (9175S;Cell Signaling Technology, Danvers, MA), and mouse monoclonal anti-human β-Actin (8H10D10; Cell Signaling Technology, Danvers, MA) antibodies were used as primary antibodies; horseradish peroxidase-labeled anti-mouse or anti-rabbit (Cell Signaling Technology, Danvers, MA) were used as the secondary antibodies. Rabbit monoclonal PD-L1 conjugated with Alexa Fluor 488 (25048, Cell Signaling Technology, Danvers, MA) was used for extracellular expression analysis via flow cytometry and immunofluorescence.

Tong S., Cinelli M.A., El-Sayed N.S., Huang H., Patel A., Silverman R.B, & Yang S. (2022). Inhibition of interferon-gamma-stimulated melanoma progression by targeting neuronal nitric oxide synthase (nNOS). Scientific Reports, 12, 1701.

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Th2 Cell Modulation in Allergy

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The antibodies of HDAC1, pSTAT6, p300, pp300, STAT3, pSTAT3, H3K4ac, RNA polymerase II, shRNA kits of p300 and STAT3 were from Santa Cruz Biotech (Santa Cruz, CA). The fluorochrome-labeled antibodies of Foxp3, CD4, IL-10, CD19 and IgE were from BD Biosciences (Franklin Lakes, NJ). The biotinylated IgE antibody was from Abcam (Cambridge, MA). Magnetic cell sorting kits were from Miltenyi Biotech (San Diego, CA). The house dust mite vaccine was from Wowu Biotech (Hangzhou, China). Clostridium butyricum was from Shenzhen Kexing Biotech (Shenzhen, China). The Der p 1 protein was from Dr. Zhijiang Liu (Shenzhen University, China). PCI-32765 was purchased from Chem Blink (Shanghai, China). Reagents for real time RT-PCR and Western blotting were from Invitrogen (Carlsbad, CA). Protein G, ChIP kit and butyrate sodium were from Sigma Aldrich (St. Louis., MO).

Liao H.Y., Tao L., Zhao J., Qin J., Zeng G.C., Cai S.W., Li Y., Zhang J, & Chen H.G. (2016). Clostridium butyricum in combination with specific immunotherapy converts antigen-specific B cells to regulatory B cells in asthmatic patients. Scientific Reports, 6, 20481.

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EGFR and STAT3 Signaling Modulation

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H1299 and HCC827 cells were treated with IFN-α (10⁴ IU/ml) or gefitinib (20 nM) alone or in combination for 48 hours, washed with cooled PBS three times, and then lysed in 50–100 µl mammalian protein extraction reagent containing a protease inhibitor cocktail for 30 minutes. The lysates were centrifuged at 12,000 g at 4°C for 10 minutes. The lysates were normalised for total protein content using a bicinchoninic acid assay, and 60 µg protein samples were loaded on SDS-PAGE gels and transferred to PVDF membranes. The membranes were probed with rabbit monoclonal antibodies against phosphorylated (p)-EGFR, EGFR, p-STAT3 (signal transducers and activators of transcription 3), STAT3, and β-actin (Santa Cruz, Dallas, TX, USA) at 4°C overnight. After washing three times in TBS-T (Tris hydrochloride buffer containing 0.1% Tween-20), the membranes were incubated with the goat polyclonal secondary antibody to rabbit for two hours at room temperature. The membranes were washed with TBS-T three times for 10 minutes and exposed to X-ray film for 5–10 minutes. The relative expression of protein was determined from the optical density ratio of the corresponding protein bands using BandScan 5.0 software (Glyko Inc., Upper Heyford, UK).

Pan C., Weng S., Duan Y., Ding L., Zhang S, & Huang J. (2016). Interferon-α reduces the gefitinib sensitivity of human non-small cell lung cancer. Contemporary Oncology, 20(4), 320-326.

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Western Blot Analysis of Cellular Signaling

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Cells and tissue homogenates (20 μg protein) were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes for antibody probing. After washing with TBST (10 mM Tris–HCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20), membranes were blocked with 5% skim milk for 2 h and then incubated with primary antibodies specific to HIF-1α, PrP^C, janus kinase 2 (JAK2), phosphorylated-JAK2, signal transducer and activator of transcription 3 (STAT3), p-STAT3, cyclin D1, c-Myc, p-NF-κB, cleaved caspase-3, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). After incubation of the membranes with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), bands were detected using enhanced chemiluminescence reagents (Amersham Biosciences, Little Chalfont, UK) in a dark room.

Han Y.S., Lee J.H., Yoon Y.M., Yun C.W., Noh H, & Lee S.H. (2016). Hypoxia-induced expression of cellular prion protein improves the therapeutic potential of mesenchymal stem cells. Cell Death & Disease, 7(10), e2395-.

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Protein Quantification and Western Blot Analysis

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Proteins were quantified using a BCA Protein Assay Kit (Solarbio, Shanghai, China), and 50 μg of the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the proteins were then electrotransferred onto a nitrocellulose membrane. Next, the membrane was treated with monoclonal antibodies against mouse ADAM9, heat shock protein 27 (HSP27), heat shock protein 70 (HSP70), proliferating cell nuclear antigen (PCNA), B cell lymphoma protein 2 (Bcl-2), Bcl-2-associated X (Bax), caspase-3, vascular endothelial growth factor (VEGF), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) (Santa Cruz). The signal was detected by employing a horseradish peroxidase detection system using diaminobenzidine (Sigma). The protein bands were quantified with the Gel-Pro Analyzer software 4.0 (Media Cybernetics Inc., Bethesda, MD) and their intensities normalized to beta-actin. Every experiment was repeated thrice [7 (link)].

Zhang Y.Y., Li S.Q., Song Y., Wang P., Song X.G., Zhu W.F, & Wang D.M. (2022). Silencing the ADAM9 Gene through CRISPR/Cas9 Protects Mice from Alcohol-Induced Acute Liver Injury. BioMed Research International, 2022, 5110161.

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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 in PANC-1 Cells

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Denaturing SDS-PAGE sample buffer was utilized for PANC-1 cell lysis through standard methods. Then, separation of protein lysates was done with 10% SDS-PAGE, and they were transferred onto nitrocellulose membranes. TBS containing 0.1% Triton X-100 and 5% nonfat milk was used overnight at 4°C to block the membranes, followed by incubation at 4°C overnight with primary antibodies of STAT3, p-STAT3, JAK, p-JAK, Bcl-2, Bax, caspase-3, caspase-9, and β-actin (Santa Cruz Biotech, Santa Cruz, CA, USA). Upon washing, the membranes were subjected to incubation with HRP-conjugated secondary antibodies for 2 h at room temperature. Detection of the signal was accomplished with the ECL reagents.

Xing H.B., Tong M.T., Wang J., Hu H., Zhai C.Y., Huang C.X, & Li D. (2018). Suppression of IL-6 Gene by shRNA Augments Gemcitabine Chemosensitization in Pancreatic Adenocarcinoma Cells. BioMed Research International, 2018, 3195025.

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Quantifying Protein Expression in Cardiac Tissue

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The BCA kit (Cwbio, Jiangsu, China) was utilized to quantify the protein isolated from cardiac tissues, which were further separated with the 12% SDS-PAGE. The separated protein was then transferred from the gel to the PVDF membrane, and mixed with 5% skim milk. Then, the membrane was introduced with the primary antibody against p-JAK-2 (1:1000, Santa Cruz Biotechnology, USA), JAK-2 (1:2000, Santa Cruz Biotechnology, USA), p-STAT-3 (1:1000, Santa Cruz Biotechnology, USA), STAT-3 (1:2000, Santa Cruz Biotechnology, USA), and β-actin (1:8000, Santa Cruz Biotechnology, USA). The secondary antibody (1:2000, SolelyBio, Beijing, China) was subsequently added to be incubated for 90 min. Finally, the ECL reagent was added to expose the bands, which were further quantified with the Image J software [16 (link)].

Chen Z., Zhou H., Huang X., Wang S., Ouyang X., Wang Y., Cao Q., Yang L., Tao Y, & Lai H. (2022). Pirfenidone attenuates cardiac hypertrophy against isoproterenol by inhibiting activation of the janus tyrosine kinase-2/signal transducer and activator of transcription 3 (JAK-2/STAT3) signaling pathway. Bioengineered, 13(5), 12772-12782.

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