Phospho stat3

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Phospho-STAT3 is a laboratory reagent used in immunoassays to detect and quantify the phosphorylated form of the STAT3 protein. STAT3 is a transcription factor that plays a key role in cellular signaling pathways. The phosphorylation of STAT3 is an important regulatory mechanism that can be studied using this product.

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21 protocols using phospho stat3

Quantifying Protein Signaling Pathways

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Western blot analysis was performed as described previously.15 (link),20 (link) The membrane containing transferred protein was incubated with the following primary antibodies overnight at 4°C: Rabbit polyclonal antibodies against signal transducer and activator of transcription 3 (STAT3; cat. no. sc-482; dilution, 1:400; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phospho (p)-STAT3 (cat. no. sc-8059; dilution, 1:500; Santa Cruz Biotechnology, Inc.), p65 (cat. no. sc-7151; dilution, 1:400; Santa Cruz Biotechnology, Inc.) and cyclooxygenase-2 (COX-2; cat. no. sc-23,983; dilution, 1:400; Santa Cruz Biotechnology, Inc.). Following washing with 0.1% Tween-20 in PBS, the membranes were then incubated with the following horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C: Goat anti-rabbit immunoglobulin G (IgG; cat. no. sc-2004; dilution, 1:2,000; Santa Cruz Biotechnology, Inc.). Proteins were detected using an enhanced chemiluminescence kit (GE Healthcare Life Sciences, Chalfont, UK). Anti-GAPDH monoclonal antibody (cat. no. ab9485; dilution, 1:2,500; Abcam, Cambridge, MA, USA) was used to ensure equal loading of protein.

You Y., Wang Q., Li H., Ma Y., Deng Y., Ye Z, & Bai F. (2019). Zoledronic acid exhibits radio-sensitizing activity in human pancreatic cancer cells via inactivation of STAT3/NF-κB signaling. OncoTargets and therapy, 12, 4323-4330.

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Adipogenic Differentiation Pathway Activation

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DHA was bought from Selleckchem (Houston, TX, USA).
Primary antibodies of STAT-3, phospho (p)-STAT-3, STAT-5, p-STAT-5, C/EBP-α, and PPAR-γ were obtained from Santa Cruz Biotechnology (Delaware, CA, USA). Primary FAS antibody was bought from BD Bioscience (San Jose, CA, USA). Primary antibodies of perilipin A and β-actin were obtained from Bio Vision (Milpitas, CA, USA) and Sigma (St. Louis, MO, USA), respectively. Enhanced chemiluminescence (ECL) reagent was bought from Advansta (Menlo Park, CA, USA).
3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin were purchased from Sigma.

Jang B. (2020). Inhibitory Effect of Dihydroartemisinin, An Active Ingredient of Artemisia annua, on Lipid Accumulation in Differentiating 3T3-L1 Preadipocytes. Journal of Korean Medicine for Obesity Research, 20(1), 1-9.

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Evaluating ECFC-Derived Vascular Formation

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After 3 and 28 days following ECFC transplantation, the ischemic thigh areas were removed and fixed with 4 % paraformaldehyde (Affymetrix, Santa Clara, CA, USA). Each tissue sample was embedded in paraffin. For histological analysis, samples were stained with hematoxylin and eosin (H&E). Immunofluorescence staining was performed using primary antibodies against mouse-specific (to confirm mouse vasculature) or human-specific (to confirm human ECFC-derived vessel formation) CD31, α-smooth muscle actin (α-SMA), human VEGF, phospho-STAT3, caspase-3, PCNA, Ki67 (Santa Cruz), human-specific CD31 (Novus Biologicals, Colorado, USA), and human nuclear antigen (HNA; Millipore) and secondary antibodies Alexa-488 and Alexa-594 (Life Technologies, Carlsbad, CA, USA). Nuclei were stained with 4′,6-diamidino-2-phenylindole phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Immunostained slides were imaged by confocal microscopy (Olympus, Japan).

Lee S.H., Lee J.H., Han Y.S., Ryu J.M., Yoon Y.M, & Han H.J. (2015). Hypoxia accelerates vascular repair of endothelial colony-forming cells on ischemic injury via STAT3-BCL3 axis. Stem Cell Research & Therapy, 6(1), 139.

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CXCL1 Signaling Pathway Activation

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Cells were seeded in 6 well plates at 250,000/well for 24 hours, serum starved for 24 hours, and stimulated with 60 ng/ml mouse recombinant CXCL1 (RnD Systems) for up to 24 hours. Cells were lysed in RIPA buffer containing protease inhibitors (Sigma cat no. P8340) and 10 μM sodium orthovanadate and sonicated. 50 μg protein were resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked in PBS containing 0.05% Tween-20 (PBS-T) containing 3% dry milk for 1 hour and immunoblotted with primary antibodies at a 1:1000 overnight in PBS-T/3% BSA. With the exception of FAK, antibodies were obtained from Cell Signaling Technology: phospho-Src (Tyr416 cat no. 6943), Src (cat no. 2123), phospho-FAK (Tyr397 cat no.8856), FAK (Santa Cruz Biotechnology, cat no. sc558), phospho-p38MAPK (Thr180/Tyr182, cat no. 4511), p38MAPK (cat no. 9212), phospho-IκB (Ser32/36, cat no. 9246), IκB (cat no. 2678), phospho-p42/44MAPK (Thr202/Tyr204, cat no. 9101), p42/44MAPK (cat no. 9102), phospho-AKT (Ser473, cat no. 4060), AKT (cat no. 2965), phospho-Stat3 (Tyr705, cat no. 9145), Stat3 (cat no. 9132). Immunoblots were washed in PBS-T 3 times for 10 minutes each and incubated with secondary anti-rabbit conjugated to horse radish peroxidase at a 1:1000 dilution in PBS-T/3% milk for 2 hours. Reactions were catalyzed with West Femto ECL substrate.

Bernard S., Myers M., Fang W.B., Zinda B., Smart C., Lambert D., Zou A., Fan F, & Cheng N. (2018). CXCL1 derived from mammary fibroblasts promotes progression of mammary lesions to invasive carcinoma through CXCR2 dependent mechanisms. Journal of mammary gland biology and neoplasia, 23(4), 249-267.

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Western Blot Analysis of HepG2 Cells

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HepG2 cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). Cell lysates (20 μg protein/lane) were loaded and separated on gradient polyacrylamide gels and then transferred to polyvinylidene difluoride membranes by electroblotting (Millipore Corp., Boston, MA, USA). Following blocking with 5% non-fat milk containing 0.3% Tween 20 for 1 h, the membranes were incubated overnight with primary antibodies at 4°C, including anti-hSulf-1 (1:250), -stat3 (1:500), -phospho-stat3 (1:500), -phospho-c-met (1:500), -bcl-2 (1:1000) and -cyclin D1 (1:500) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were washed three times with Tris-buffered saline containing Tween 20 and membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (R&D Systems China Co., Ltd., Shanghai, China) at 4°C for 1 h. Subsequently, membranes were exposed to enhanced chemiluminescent reagents for detection of protein bands. β-actin was used as an internal control.

LIU L., DING F., CHEN J., WANG B, & LIU Z. (2014). hSulf-1 inhibits cell proliferation and migration and promotes apoptosis by suppressing stat3 signaling in hepatocellular carcinoma. Oncology Letters, 7(4), 963-969.

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Western Blot Analysis of Signaling Proteins

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Tissues or cells were mechanically homogenized in ice-cold lysis buffer and centrifuged for 10 minutes at 4 °C and 14,000 g for tissues or 8,000 g for cells. The resulting supernatant fraction was separated by SDS-PAGE and immunoblotted with antibodies against phospho-AKT (S473; 1:1000), total AKT (1:1000), total JAK2 (1:1000), tubulin (1:1000), phospho-STAT3 (Y705, 1:2000), total STAT3 (1:1000), phospho-STAT5 (Y694, 1:1000), total STAT5 (1:1000), phospho-NF-κB p65 (Y694, 1:1000), total NF-κB p65 (1:1000), phospho-JNK (T183/Y185, 1:500, Santa Cruz Biotechnology) or total JNK1 (1:500, Santa Cruz Biotechnology) or GAPDH (1:5000). Antibodies were purchased from Cell Signaling Technology unless otherwise stated. Protein band intensity was quantified by ImageJ software.

Desai H.R., Sivasubramaniyam T., Revelo X.S., Schroer S.A., Luk C.T., Rikkala P.R., Metherel A.H., Dodington D.W., Park Y.J., Kim M.J., Rapps J.A., Besla R., Robbins C.S., Wagner K.U., Bazinet R.P., Winer D.A, & Woo M. (2017). Macrophage JAK2 deficiency protects against high-fat diet-induced inflammation. Scientific Reports, 7, 7653.

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Immunohistochemical Analysis of Signaling Pathways

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Immunohistochemistry was performed in paraffin-embedded serial sections using phospho-STAT3 (Santa Cruz), STAT3, phospho-Akt, Akt, phospho-ERK, ERK (Cell Signaling), and Ki67 (Biocare Medical, Concord, CA) antibodies, as described [30 (link)].

Neiva K.G., Warner K.A., Campos M.S., Zhang Z., Moren J., Danciu T.E, & Nör J.E. (2014). Endothelial cell-derived interleukin-6 regulates tumor growth. BMC Cancer, 14, 99.

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Adipose Tissue Protein Profiling

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Hypothalami, epididymal fats, and liver tissues (n = 6/group) were isolated, and proteins extracted using the commercial Tissue Protein Extraction Reagents (T-PER, Pierce Biotechnology, Rockford, IL, USA). Equal amounts of proteins were resolved by SDS-PAGE, before being transferred to the polyvinylidine fluoride membrane. The membranes were blocked with 5% nonfat milk and incubated with primary antibodies, horseradish peroxidase-conjugated IgG, and enhanced chemiluminescence Western blotting reagents. The visualized signals were quantitated by a densitor meter. Primary antibodies used were: Akt, phospho-Akt, signal transducers and activators of transcription 3 (STAT3), phospho-STAT3, STAT6, phospho-STAT6, PRDM16, PGC-1α, UCP-1, CD68, CD206, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Lin S.Y., Yang C.P., Wang Y.Y., Hsiao C.W., Chen W.Y., Liao S.L., Lo Y.L., Chang Y.H., Hong C.J, & Chen C.J. (2020). Interleukin-4 Improves Metabolic Abnormalities in Leptin-Deficient and High-Fat Diet Mice. International Journal of Molecular Sciences, 21(12), 4451.

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Western Blot Analysis of Signaling Proteins

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Western blot analyses were performed following a standard procedure. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with the antibodies raised against phospho-S33/S37/T41-BCATENIN (Cell Signaling Technology [CST], #9561), phospho-S465/467-SMAD2 (CST, #3101), SMAD2/3 (CST, #3102), p-ERK (CST, #4370), ERK (CST, #9102), BCATENIN (BD Biosciences, C19220-050), phospho-STAT3 (Santa Cruz Biotechnology, sc-8059), and STAT3 (Santa Cruz, sc-482). Protein bands were visualized using ECL Prime Western Blotting Detection Reagent (GE Healthcare).

Illich D.J., Zhang M., Ursu A., Osorno R., Kim K.P., Yoon J., Araúzo-Bravo M.J., Wu G., Esch D., Sabour D., Colby D., Grassme K.S., Chen J., Greber B., Höing S., Herzog W., Ziegler S., Chambers I., Gao S., Waldmann H, & Schöler H.R. (2016). Distinct Signaling Requirements for the Establishment of ESC Pluripotency in Late-Stage EpiSCs. Cell Reports, 15(4), 787-800.

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Synergistic Anticancer Effects of Selinexor and Sorafenib

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Selinexor was provided by Karyopharm Therapeutics (Newton, MA, USA). Sorafenib was purchased from Selleckchem (Houston, TX, USA). Their molecular structures are shown in Online Supplementary Figure S1. The antibodies against human phosphorylated (p)-p44/42 MAPK (ERK1/2)(Thr202/Tyr204), phospho-AKT(Ser473), phospho-FLT3(Tyr589/591), phospho-S6K(Ser240/244), AKT, S6K, Bcl-xL, C/EBPα, PU.1, STAT3, c-Myc and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA), against Bcl-2 from Dako (Carpinteria, CA, USA), against phospho-STAT5 A/B from Upstate (Lake Placid, NY, USA), against total STAT5A/B from R&D Systems Inc. (Minneapolis, MN, USA), against ERK2, FLT3, p53, IκB alpha, phospho-STAT3, and Mcl-1 from Santa Cruz Biotechnology (Santa Cruz, CA, USA), against Bim and Puma from CalBiochem (San Diego, CA, USA), against HIF1α from BD Biosciences (San Diego, CA, USA), and against phospho-IκB alpha (ser32/36) from Novus (Littleton, CO, USA). The anti-luciferase antibody was purchased from Promega (Madison, WI, USA).

Zhang W., Ly C., Ishizawa J., Mu H., Ruvolo V., Shacham S., Daver N, & Andreeff M. (2018). Combinatorial targeting of XPO1 and FLT3 exerts synergistic anti-leukemia effects through induction of differentiation and apoptosis in FLT3-mutated acute myeloid leukemias: from concept to clinical trial. Haematologica, 103(10), 1642-1653.

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