Rabbit anti phospho stat3

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-phospho-STAT3 is a primary antibody that specifically recognizes the phosphorylated form of STAT3 (Signal Transducer and Activator of Transcription 3) protein. It is designed for use in various immunoassay applications, such as Western blotting, to detect and measure the levels of activated STAT3 in biological samples.

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Close spelling variants of this product

STAT3 (1075 citations) Anti-STAT3 (536 citations) Phospho-STAT3 (345 citations) Anti-phospho-STAT3 (149 citations) Rabbit anti-STAT3 (53 citations) Rabbit anti-phospho-STAT3 (Tyr705) (16 citations) Rabbit monoclonal anti-phospho-STAT3 (Tyr705) (5 citations) Rabbit anti-phospho-STAT3 (Tyr705) (D3A7) (4 citations) Rabbit anti-phospho-STAT3 (Tyr 705) antibody (3 citations) Rabbit anti‐phospho‐Stat3 antibody (2 citations)

25 protocols using rabbit anti phospho stat3

Kinase Assay of STAT3 and Chk1

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Kinase assays were performed using recombinant full-length human GST-STAT3 (SignalChem C47-10H) together with recombinant full-length human His-Chk1 (SignalChem S54-54G), in kinase buffer (20 mM Hepes pH 7.4, 10 mM MgCl₂, 10 mM MgCl₂, 1 mM EGTA, 0.1 mM Na3VO4, 0.5 mM NaF, 50 mM β-glycerophosphatase, 50 mM DTT) in the presence of 50 μM ATP and 5 μCi [³²P]-ATP (EasyTide). The kinase reaction proceeded for 45 min at 30 °C, and was stopped by the addition of NuPAGE™ LDS Sample Buffer (4X) (Invitrogen NP0007), boiled for 10 min at 85°C, and analyzed by electrophoresis on 4-12% SDS-polyacrylamide gel. Gels were stained with Coomassie Brilliant Blue (CBB), then fixed, dried and subjected to autoradiography. A non-radioactive Kinase Assay was performed without cold ATP. Kinase products were analyzed by electrophoresis on 4-12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes, which were further stained Ponceau S. Immunoblotting was analyzed with rabbit anti-phospho-STAT3 (Tyr705, Cell Signaling Technology Cat# 2577), rabbit anti-phospho-STAT3 (Ser727, Cell Signaling Technology Cat# 9134) and rabbit anti-phospho-Chk1 (Ser296, Cell Signaling Technology Cat# 2349). Images were quantified and analyzed using ImageJ software (ImageJ, RRID:SCR_003070).

Zhou L., Pei X., Zhang Y., Ning Y., Li L., Hu X., Chalasani S.L., Sharma K., Nkwocha J., Yu J., Bandyopadhyay D., Sebti S.M, & Grant S. (2022). Chk1 inhibition potently blocks STAT3 tyrosine705 phosphorylation, DNA binding activity, and activation of downstream targets in human multiple myeloma cells. Molecular cancer research : MCR, 20(3), 456-467.

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Immunofluorescence and Immunoblotting Analysis

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Immunofluorescences (IF) and immunoblots (WB) were performed with the following primary antibodies: anti-rabbit S100A4 (1:500-IF, 1:1000-WB, Millipore), anti-rabbit mTOR and phospho-mTOR (1:100-WB, Cell Signaling), anti-mouse SQSTM1/p62 (1:1000-WB, Abcam), anti-rabbit NF-kB and phospho-NF-kB (1:1000-WB, Cell Signaling), anti-rabbit STAT3 (1:1000-WB, Cell Signaling), anti-rabbit phospho-STAT3 (1:2000-WB, Cell Signaling), anti-rabbit α-SMA (1:1000-WB, 1:500-IF, GeneTex), anti-rabbit N-cadherin (1:1000-WB, GeneTex), anti-mouse glial fibrillary acidic protein (GFAP) (1:500-IF, 1:1000-WB, Cell Signaling), anti-rabbit β-III Tubulin (1:500-IF, Cell Signaling), anti-mouse MyoG (1:200-WB, Hybridoma Bank, USA), anti-mouse platelet-derived growth factor receptor β (PDGFR-β) (1:250-WB, 1:500-IF, Santa Cruz), and anti-mouse GAPDH (1:10000-WB, Calbiochem). Secondary immunoblot antibodies for WB were anti-rabbit (1:2500) and anti-mouse (1:5000) IgG peroxidase-conjugated from Bio-Rad Laboratories (Hercules, CA, USA). Secondary fluorescent antibodies for IF were Alexa-Flour 488-Donkey anti-rabbit (1:200) and Cy3-Donkey anti-mouse (1:200) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). DAPI was used to stain nuclei (1:1000, Thermo Fisher Scientific, Waltham, MA, USA).

Milani M., Mammarella E., Rossi S., Miele C., Lattante S., Sabatelli M., Cozzolino M., D’Ambrosi N, & Apolloni S. (2021). Targeting S100A4 with niclosamide attenuates inflammatory and profibrotic pathways in models of amyotrophic lateral sclerosis. Journal of Neuroinflammation, 18, 132.

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Western Blot Analysis of Hippocampal Proteins

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Mouse brains were quickly removed, and the hippocampi were immediately dissected and frozen. Tissue was prepared using a hypertonic lysis buffer containing protease inhibitors as previously described [68 (link)]. The primary antibodies used were anti-rabbit phospho-IKKα/β (1:1000 dilution; Cell Signaling, Alcobendas, Spain, 2861), anti-rabbit IKK α/β (1:1000 dilution; Santa Cruz Biotechnology, Heidelberg, Germany, sc-7607), anti-rabbit phospho-STAT3 (1:1000 dilution; Cell Signaling, 9145) and anti-mouse STAT3 (1:1000 dilution; Santa Cruz Biotechnology, sc-8019). Anti-mouse α-tubulin (1:2000 dilution; Santa Cruz Biotechnology, sc-32293) was used as loading control. Anti-rabbit HRP (1:5000 dilution; Jackson ImmunoResearch Laboratories, Baltimore, USA, 111-035-144) and anti-mouse HRP (1:5000 dilution; Jackson ImmunoResearch Laboratories, 115-035-003) were used as secondary antibodies. Protein band quantification was carried out by band densitometry analysis with the NIH ImageJ Software (Version 1.50i, https://imagej.nih.gov/ij/, accessed on 1 March 2022). To ensure equal loads, the integrated density obtained for each band was expressed as a relative value with respect to the appropriate housekeeping loading control.

Calvo B., Fernandez M., Rincon M, & Tranque P. (2022). GSK3β Inhibition by Phosphorylation at Ser389 Controls Neuroinflammation. International Journal of Molecular Sciences, 24(1), 337.

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THSG Modulates JAK2/STAT3 Signaling

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After the cells were treated with 0, 1, 10, and 100 μM THSG in PluriSTEMTM ES/iPS Medium (Merk) for 3 h, protein extraction and Western blotting were performed as described previously.¹³ (link) The resolved proteins were then incubated with rabbit anti-JAK2, rabbit anti-phospho-JAK2, rabbit anti-STAT3, rabbit anti-phospho-STAT3 (Cell Signaling Technology, Beverly, MA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Merk, Darmstadt, Germany) at 4 °C overnight. Signals were detected using the LumiFlas™ Femto Chemiluminescent Substrate, HRP kit (ENERGENESIS BIOMEDICAL, Taipei, Taiwan). Images of the Western blots were visualized and analyzed using the Chemi-Doc™ XRS + System (Bio-Rad Laboratories, Hercules, CA, USA).

Huang Y.W., Lin C.Y., Chin Y.T., Kuo P.J., Wu Y., Weng I.T., Chen T.Y., Wang H.H., Huang H.M., Hsiung C.N, & Lee S.Y. (2020). 2,3,5,4′-tetrahydroxystilbene-2-O-b-D-glucoside triggers the pluripotent-like possibility of dental pulp stem cells by activating the JAK2/STAT3 axis: Preliminary observations. Journal of Dental Sciences, 16(2), 599-607.

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Western Blot Analysis of Phospho-STAT3 and STAT3

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The protein levels of phospho-STAT3 and STAT3 were detected by Western blotting as previously described (Li et al., 2020 (link)). The primary antibodies were as follows: rabbit anti-phospho-STAT3 (1:2000, Cell Signaling Technology), mouse anti-STAT3 (1:1,000, Cell Signaling Technology) and mouse anti-β-actin (1:5,000, Sigma). Chemiluminescence was detected by ChemiDoc^TM Touch Imaging System (Bio-Rad).

Li S., Dou B., Shu S., Wei L., Zhu S., Ke Z, & Wang Z. (2022). Suppressing NK Cells by Astragaloside IV Protects Against Acute Ischemic Stroke in Mice Via Inhibiting STAT3. Frontiers in Pharmacology, 12, 802047.

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Investigating Cytokine and Signaling Pathways in EMT

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Human recombinant IL-6 and IL-6 neutralizing antibody were purchased from R&D Systems Inc. (Minneapolis, MN, USA). STAT3 inhibitor Stattic was purchased from Sigma-Aldrich Inc. (St Louis, MO, USA), JAK2 inhibitor AG490 was purchased from Abcam (Cambridge, MA, USA).
Antibodies used for western blotting were mouse anti-E-cadherin (Invitrogen, Carlsbad, CA, USA), mouse anti-vimentin (BD Bioscience, San Jose, CA, USA), rabbit anti-phospho-JAK2 (Cell Signaling Technology, Beverly, MA, USA), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti-phospho-STAT3 (Cell Signaling Technology), mouse anti-α-SMA (Abcam), rabbit anti-MMP2 (Cell Signaling Technology), mouse anti-VEGF (Cell Signaling Technology), rabbit anti-TWIST (Cell Signaling Technology), mouse anti-β-actin (Sigma-Aldrich). The secondary antibodies coupled to HRP were purchased from ZSGB-BIO (Beijing, China)

Wang L., Cao L., Wang H., Liu B., Zhang Q., Meng Z., Wu X., Zhou Q, & Xu K. (2017). Cancer-associated fibroblasts enhance metastatic potential of lung cancer cells through IL-6/STAT3 signaling pathway. Oncotarget, 8(44), 76116-76128.

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Immunohistochemical Profiling of Cell Markers

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Immunohistochemistry was performed as previously described^{65 (link)}. The following primary antibodies were used: rabbit anti-Cleaved caspase 3 (1:200; Cell Signalling #9661), mouse anti-CD45 (1:20, BDPharmigen #550539), rabbit anti-CK19 (1:1000; Abcam #ab133496), goat anti-CPA1 (1:300, RD Systems #AF2765), mouse anti–Ki-67 (1:400; BDPharmigen #550609), mouse anti-MUC5AC (1:200; Cell Marque #292M-95), rabbit anti-p65 (C-20) (1:200, Santa Cruz #sc-372), rabbit anti-phospho-STAT3 (Y705) (1:100, Cell Signaling #9145).

Hidalgo-Sastre A., Lubeseder-Martellato C., Engleitner T., Steiger K., Zhong S., Desztics J., Öllinger R., Rad R., Schmid R.M., Hermeking H., Siveke J.T, & von Figura G. (2020). Mir34a constrains pancreatic carcinogenesis. Scientific Reports, 10, 9654.

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Western Blot Analysis of Inflammatory Signaling

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Synovial tissues from the right forepaw joints and treated macrophages were lysed in RIPA buffer (Solarbio, Beijing, China, Cat#R0020) with phenylmethylsulfonyl fluoride (Solarbio, Cat#P0100) and protein phosphatase inhibitor (Solarbio, Cat#P1260). Protein samples were separated by SDS-PAGE. Separated proteins were transferred onto polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA, Cat#IPVH00010). For specific protein detection, the following primary antibodies were used: rabbit anti-STAT3 (Cell Signaling Technology, Danvers MA, USA, Cat#9139S), rabbit anti-Phospho-STAT3 (Cell Signaling Technology, Cat#9145S), rabbit anti-NLRP3 (Invitrogen, Waltham, MA, USA, Cat# PA5-79740), mouse anti-HIF-1α (Santa Cruze, Dallas, TX, USA, Cat#13515), and rabbit anti-GAPDH (Abcam, Cambridge, UK, Cat# 8245). Lastly, the Western blotting bands were analyzed with Image J Software.

Wu D., Xu J., Jiao W., Liu L., Yu J., Zhang M, & Chen G. (2023). Suppression of Macrophage Activation by Sodium Danshensu via HIF-1α/STAT3/NLRP3 Pathway Ameliorated Collagen-Induced Arthritis in Mice. Molecules, 28(4), 1551.

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Salmonella Strains and Reagents

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All bacterial strains used in this study were derived from the Salmonella enterica serovar Typhimurium strain SL1344 [47 (link)] or Salmonella enterica serovar Typhi strain ISP2825 [48 (link)] and are listed in S2 Table. Plasmids used in this study are listed in S3 Table and have either been described before or were constructed as part of this study using standard recombinant DNA techniques. S. Typhimurium and S. Typhi were grown under conditions that increase expression of the SPI-1 T3SS [49 (link)]. Briefly, a 1:20 dilution of a bacterial overnight cultures were grown at 37°C in L-broth containing 0.3 M NaCl until an OD₆₀₀ = 0.9 prior to their use in infections. Antibodies and other reagents were purchased from the indicated companies: rabbit-anti-TTP (Abcam); rabbit-anti-Salmonella O Group B Antiserum (Becton Dickson); rabbit-anti- Phospho-STAT3 (Ser727), rabbit-anti-Phospho-STAT3 (Tyr705), Erk (Thr202, Tyr204), JNK (Thr183, Tyr185), P38 (Thr202, Tyr204) and I-kBα (Cell Signaling Technology); mouse-anti-SerpinB3 (Santa Cruz Biotechnology); mouse-anti-tubulin (Sigma-Aldrich); secondary antibodies (Molecular Probes); 49,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich).

Hannemann S, & Galán J.E. (2017). Salmonella enterica serovar-specific transcriptional reprogramming of infected cells. PLoS Pathogens, 13(7), e1006532.

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Immunoblotting Antibody Reagents

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The following antibodies, from the indicated sources, were used: rabbit anti-phospho-STAT3 (#9145), rabbit anti-STAT3 (#8768), rabbit anti-phospho-JAK1 (#74129), rabbit anti-myc (#9145), alpha-tubulin (DM1A, #3873) all from Cell Signaling Technology; mouse anti HA11 (#901514) and anti-Tuj1 (both from Biolegend); mouse anti-myc 9E10 (UPenn Cell Center); rabbit anti-GFP #A11122 (Invitrogen); mouse anti-JAK1 #610231 (BD Transduction Labs), mouse-anti NeuN #MAB377 (Millipore Sigma), Calnexin #10286 (Abcam). Unless otherwise noted, all chemicals were from ThermoFisher Biosciences and were of the highest reagent grade available.

Hernandez L.M., Montersino A., Niu J., Guo S., Faezov B., Sanders S.S., Dunbrack R.L, & Thomas G.M. (2023). Palmitoylation-dependent control of JAK1 kinase signaling governs responses to neuropoietic cytokines and survival in DRG neurons. The Journal of Biological Chemistry, 299(8), 104965.

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