Stat3 124h6

Manufactured by Cell Signaling Technology

Sourced in United States

STAT3 (124H6) is a mouse monoclonal antibody that recognizes STAT3, a member of the STAT family of transcription factors. STAT3 plays a critical role in cellular signaling pathways and is involved in various biological processes, including cell growth, differentiation, and immune response. The STAT3 (124H6) antibody can be used in research applications to detect and study the expression and localization of STAT3 in biological samples.

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Lab products found in correlation

P stat3 ser727, by Cell Signaling Technology (1 mentions) Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions) Cd11b m1 70, by Thermo Fisher Scientific (1 mentions) Ebio17b7, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by BioLegend (1 mentions) F4 80 bm8, by BioLegend (1 mentions) Jes5 16e3, by Thermo Fisher Scientific (1 mentions) Rorγt afkjs 9, by Thermo Fisher Scientific (1 mentions) Rm4 5, by BioLegend (1 mentions) Ifn γ xmg1.2, by BioLegend (1 mentions) Nsun2, by Proteintech (1 mentions) β actin, by Merck Group (1 mentions) Fluorescence microscope, by Nikon (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Tbs blocking buffer, by LI COR (1 mentions) Phospho stat3 tyr705 d3a7, by Cell Signaling Technology (1 mentions) Myosin iia, by Merck Group (1 mentions) Odyssey fc imaging system, by LI COR (1 mentions) Irdye 680rd goat anti mouse igg, by LI COR (1 mentions) Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)

9 protocols using stat3 124h6

Molecular Signaling Pathway Analysis

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All utilized reagents were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Primary antibodies against GAPDH (FL-335), p-CaMKII α (22B1), CaMKII (M-176), AMPK α1/2 (H-300), SOD2 (FL-222), p21 (C-19), peroxidase-conjugated secondary antibodies for Western blot analysis, and FITC-conjugated antibodies for immunofluorescence study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). STAT3 (124H6) and p-STAT3 (Ser727) were purchased from Cell Signaling Technology (Danvers, MA, USA).
The CellROX® Oxidative Stress Reagent kit (C10443) was purchased from Thermo Fisher Scientific, Life Technologies Italia (Monza, Italy).

Vacante F., Senesi P., Montesano A., Frigerio A., Luzi L, & Terruzzi I. (2018). L-Carnitine: An Antioxidant Remedy for the Survival of Cardiomyocytes under Hyperglycemic Condition. Journal of Diabetes Research, 2018, 4028297.

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Western Blot Analysis of Apoptosis Markers

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At the end of the desired treatment, total cell lysates preparation and Western blot analyses were performed according to the procedure described before [38 (link)]. Puma (B-6, sc-377015) and Noxa (114c307) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Bax, p53 (1c12), phospho-p53 (Ser15), cleaved Caspase-3 (ASP175), Phospho-STAT3 (Tyr705), Phospho-STAT3 (Ser727), and STAT3 (124H6) were supplied by Cell Signaling Technology (Danvers, MA, USA).

Zhang X., Qiao Z., Guan B., Wang F., Shen X., Shu H., Shan Y., Cong Y., Xing S, & Yu Z. (2024). Fluacrypyrim Protects Hematopoietic Stem and Progenitor Cells against Irradiation via Apoptosis Prevention. Molecules, 29(4), 816.

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Comprehensive Immune Cell Profiling

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Anti-mouse phosphor-STAT3 (EP2147Y, Abcam), phosphor-NF-κB (pp65) (sc-52401, Santa), phosphor-JNK (pJNK)(9H8), phosphor-ERK (pERK)(197G2), STAT3 (124H6, Cell Signaling), NF-κBp65 (sc8008, Santa), JNK (SC), ERK(137F5), and β-actin (sc-47778, Santa) were purchased. FITC-, PE-, APC- APC/cy7-, PerCP/Cy5.5-, or PE/cy7-conjugated anti-mouse CD4 (RM4-5, Biolegend), CD8 (ZUT270.5, Biolegend), CD45.1 (30-F11, Biolegend), MHCII (I-A/I-E, M5/114.115.2, Biolegend), CD11c (MCA1441GA, Biolegend), CD11b (M1/70, eBioscience), F4/80 (BM8, Biolegend), CD206 (CO68C2), Gr-1(RB6-8C5, eBioscience), IL-10 (JES5-16E3, eBioscience), Foxp3 (MF23, eBioscience), IL-22 (140301, R&D system), IFNγ (XMG1.2, Biolegend), RORγt (AFKJS-9, eBioscience), IL-17A (eBio17B7, eBioscience), and CD3ζ (H146-968, Abcam) antibodies were purchased. Anti- REG3γ (PA517, Thermo) and anti-MUC2 (H300, Santa) were also purchased. FITC-, PE-, APC- APC/cy7-, PerCP/Cy5.5-, or PE/cy7-conjugated isotypic controls were from Biolegend. RPMI1640, DMEM, fetal bovine serum (FBS), and antibiotics were obtained from HyClone. Alexa fluor 488-conjugated goat anti-rabbit IgGH&L and Alexa fluor 594-conjugated goat anti-rabbit IgGH&L (Abcam) were also purchased. 7-AAD was from Abcam.
Lactobacillus reuteri (ATCC PTA 4659) was a gift of BioGaaia, Sweden.

Huang Y., Qi H., Zhang Z., Wang E., Yun H., Yan H., Su X., Liu Y., Tang Z., Gao Y., Shang W., Zhou J., Wang T., Che Y., Zhang Y, & Yang R. (2017). Gut REG3γ-Associated Lactobacillus Induces Anti-inflammatory Macrophages to Maintain Adipose Tissue Homeostasis. Frontiers in Immunology, 8, 1063.

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Western Blot Analysis of Epigenetic Regulators

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Whole‐cell lysates were prepared using modified radioimmunoprecipitation assay (RIPA) buffer as described previously [28 (link)]. The following primary antibodies were used for Western blotting at 1 : 1000 dilution: NSUN5 (H‐10) (Santa Cruz Biotechnology, Dallas, TX, USA; Cat# sc‐376 147, RRID: AB_10989978), NSUN2 (Proteintech, Rosemont, IL, USA; Cat# 20854‐1‐AP, RRID: AB_10693629), STAT3 (124H6) (Cell Signaling Technology, Danverss, MA, USA; Cat#9139, RRID: AB_331757), β‐Actin (Sigma‐Aldrich, Oakville, ON, Canada; Cat# A5441, RRID: AB_476744), and Tubulin (Abcam, Toronto, ON, Canada, ab59680).

Zhou J., Kong Y.S., Vincent K.M., Dieters‐Castator D., Bukhari A.B., Glubrecht D., Liu R., Quilty D., Findlay S.D., Huang X., Xu Z., Yang R.Z., Zhang L., Tang E., Lajoie G., Eisenstat D.D., Gamper A.M., Fahlman R., Godbout R., Postovit L, & Fu Y. (2023). RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma. Molecular Oncology, 17(9), 1763-1783.

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Immunofluorescence Assay for STAT3 Activation

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The cells (HEp-2 and PANC-1) were grown on 4 Permanox chambers slides. SBT-100 antibody was added overnight (1 to 10 dilutions in media) and the slides were kept at 37 °C. No SBT-100 antibody was added to the negative control samples. The following day, cells were stimulated with IL-6 (Peprotech, Cranbury, NJ, USA, 100 ng/mL) for 15 min. After stimulation, the chamber slides were immediately fixed in ice-cold 100% methanol for 10 min at 20°C. The slides were dried and proceeded with the previously mentioned IFA steps. Slides were blocked with 3% BSA in PBS at room temperature for 1 h, then the primary antibody, STAT3 (124H6, Cell Signaling Technology) overnight at 4 °C. The secondary antibody anti-mouse IgG (H and L), Alexa Fluor 488 (Cell Signaling Technology) was added for 1 h at room temperature. Lastly, the chamber slides were washed and mounted with our mounting media and viewed in the Nikon Fluorescence microscope.

Singh S., Murillo G., Richner J., Singh S.P., Berleth E., Kumar V., Mehta R., Ramiya V, & Parihar A.S. (2022). A Broad-Based Characterization of a Cell-Penetrating, Single Domain Camelid Bi-Specific Antibody Monomer That Targets STAT3 and KRAS Dependent Cancers. International Journal of Molecular Sciences, 23(14), 7565.

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Kidney Protein Extraction and Quantification

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Kidney tissue was extracted in RIPA buffer, 50mM Tris-HCl (pH 7.4), 1% Triton-x-100, 0.25% Na-deoxycholate, 200mM NaCl, 1mM EDTA, protease inhibitor cocktail (#P8340; Sigma-Aldrich), 1mM Na3VO4 (1:200), 1mM NaF (1:200), using TissueLyser LT (Qiagen; Hilden, Germany). Total protein contend was determined with BCA Protein Assay Kit (#23225; Pierce / Thermo Fisher Scientific). Total kidney lysates, 35μg per lane were separated on 10% SDS-PAGE gels using Tris/glycine buffer. After transfer onto nitrocellulose membrane, Amersham Protran (#10600003; Sigma-Aldrich), membranes were blocked using 5% BSA in TBST with 50% (v/v) Intercept (TBS) blocking buffer (#927-60001; LI-COR Biosciences, Bad Homburg, Germany) and developed using primary antisera, phospho-STAT3 (Tyr705) (D3A7) (#9145), STAT3 (124H6) (#9139), both from Cell Signaling Technology, and myosin IIA (#M8064; Sigma-Aldrich). Secondary antisera used were, IRDye 800CW goat anti-rabbit-IgG (#926-32211), IRDye 680RD goat anti-mouse-IgG (#926-68070) and protein band intensities quantified based on fluorescence detection using the Odyssey Fc Imaging System, all from LI-COR Biosciences.

Fox J.C., Hahnenstein S.T., Hassan F., Grund A., Haffner D, & Ziegler W.H. (2024). Defects of renal tubular homeostasis and cystogenesis in the Pkhd1 knockout. iScience, 27(4), 109487.

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Western Blot Analysis of Cell Signaling

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Total protein cell extracts were obtained by lysing the cells with RIPA buffer supplemented with 1x protease inhibitors (PI) (Roche, Milan, Italy). Proteins were quantified using the BCA method (Pierce, ThermoFisher Scientific), and then 30–50 µg of proteins were loaded on 8–12% polyacrylamide gels for SDS-PAGE. After the separation, the proteins were transferred on a nitrocellulose membrane (Amersham, Merck,) which was probed over-night at 4 °C with specific antibodies diluted in 1–3% skimmed milk-PBS-0.1% Tween solution: TNFR1 (H-5, Santa Cruz Biotechnologies, DBA, Milan, Italy), STAT3 (124H6, Cell Signaling Technologies, Euroclone, Milan, Italy), pSTAT3 (Y705, Cell Signaling Technologies), HSP70 (C92F3A-5, Santa Cruz Biotechnologies), TEL2 (E-1, Santa Cruz Biotechnologies), GAPDH (6C5, Santa Cruz Biotechnologies), H3 (Abcam), pIκBα (Ser32/36, Santa Cruz Biotechnologies). Detection was performed with ECL Select Reagent (GE Healthcare, Cytiva) using UVITec Alliance LD2 (UVITec Cambridge, UK) imaging system.

Meškytė E.M., Pezzè L., Bartolomei L., Forcato M., Bocci I.A., Bertalot G., Barbareschi M., Oliveira-Ferrer L., Bisio A., Bicciato S., Baltriukienė D, & Ciribilli Y. (2023). ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis. Cell Death & Disease, 14(4), 263.

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Magnolol Induces Apoptosis Signaling

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Magnolol was purchased from Wuhan ChemFaces Biochemical Co., Ltd. (Wuhan, Hubei, China). MTT and dimethyl sulfoxide (DMSO) were both obtained from Sigma Chemical Co. (St. Louis, MO, USA). Roswell Park Memorial Institute 1640 (RPMI 1640) medium, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin (PS) were purchased from GIBCO^®/Invitrogen Life Technologies (Carlsbad, CA, USA). Primary antibodies against Death receptor 4 (DR4) (D9S1R) (#42533, Cell Signaling Technology, Danvers, MA, USA), Death receptor 5 (DR5) (D4E9) XP^® (#8074, Cell Signaling Technology), MMP-9 (PA5-13199, Invitrogen, Waltham, MA, USA), uPA (GTX79597, GeneTex, Irvine, CA, USA), PKCδ (Thr505) (#9374, Cell Signaling Technology), PKCδ (D10E2) (#9616, Cell Signaling Technology), STAT3 (Tyr705) (D3A7) XP^® (#9145, Cell Signaling Technology), STAT3 (124H6) (#9139, Cell Signaling Technology), Myeloid-cell leukemia-1 (MCL-1) (D35A5) (#5453, Cell Signaling Technology), and β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA) for Western blotting were all purchased from different companies as mentioned. Secondary antibodies, peroxidase affiniPure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG were purchased from Jackson Immunoresearch Laboratories Inc. (West Grove, PA, USA).

Yueh P.F., Lee Y.H., Fu C.Y., Tung C.B., Hsu F.T, & Lan K.L. (2021). Magnolol Induces the Extrinsic/Intrinsic Apoptosis Pathways and Inhibits STAT3 Signaling-Mediated Invasion of Glioblastoma Cells. Life, 11(12), 1399.

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Immunoblotting of Melanocyte Proteins

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Whole-cell extracts were lysed using Pierce RIPA buffer (Thermo Scientific) or NP-40 buffer supplemented with 1x Halt protease inhibitor cocktail (ThermoFisher) and 1x Halt phosphatase inhibitor cocktail (ThermoFisher). The concentration of cell extracts was measured by the Bio-Rad Protein Assay following manufacturer’s protocol. The same amounts of protein extracts were separated on the 4%−20% Mini-Protean TGX gel system (Bio-Rad) and transferred to PVDF (0.45 μm pore size, Millipore) membranes. Membranes were then incubated with the following primary antibodies: STAT1 (D1K9Y, 1:1000, Cell Signaling Technology), p-STAT1 (Y701, 58D6, 1:1000, Cell Signaling Technology), STAT3 (124H6, 1:1000, Cell Signaling Technology), p-STAT3 (Y705, D3A7, 1:2000, Cell Signaling Technology), IRF1 (D5E4, 1:1000, Cell Signaling Technology), TYR (PEP7h, 1:5000; gift from Vincent J. Hearing), TYRP1 (PEP1, 1:5000; gift from VJH), DCT (PEP8, 1:5000; gift from VJH), MITF (D5G7V, 1:1000, Cell Signaling Technology), PMEL/gp100 (EP4863(2), 1:1000, Abcam), and GAPDH-HRP (D16H11, 1:1000, Cell Signaling Technology). The secondary antibodies used for detection were HRP-conjugated goat anti-mouse and goat anti-rabbit IgG (1:5000, ThermoFisher). Band intensities of TIFF images were quantified by using Image J software.

Mo X., Raza Kazmi H., Preston-Alp S., Zhou B, & Raza Zaidi M. (2022). Interferon-gamma Induces Melanogenesis Via Post-Translational Regulation of Tyrosinase. Pigment cell & melanoma research, 35(3), 342-355.

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