Phospho stat6

Manufactured by Santa Cruz Biotechnology

Sourced in China, United States, Germany

Phospho-STAT6 is a lab equipment product that is used to detect and measure the phosphorylation status of the STAT6 protein. STAT6 is a transcription factor that plays a critical role in the regulation of various cellular processes. The Phospho-STAT6 product allows researchers to monitor the activation and signaling of the STAT6 protein, which is important for understanding its function in different biological systems.

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STAT6 (28 citations) Anti-phospho-STAT6 (SC-11762X (2 citations)

5 protocols using phospho stat6

Protein Expression Analysis in Mouse Lung

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Total proteins were extracted with RIPA lysis containing protease inhibitors from mouse lung tissues and Raw264.7 cells. And, protein samples were separated through 10–13% SDS-PAGE, transferred to PVDF membranes. The membranes were incubated with primary antibodies: total-STAT1 (1:1000, PTM-5754, PTM Bio, Hangzhou, China), Phospho-STAT1 (1:1000, 340797, ZenBio, Chengdu, China), total-STAT6 (1:1000, 380957, ZenBio), Phospho-STAT6 (1:500, sc-136019, Santa Cruz), iNOS (1:1000, ab178945, Abcam), Arg1 (1:1000, #93668S, Cell signaling technology), Caspase1 (1:500, sc-392736, Santa Cruz), MT1/2 (1:500, sc-398788, Santa Cruz), β-Tublin (1:5000, M20005, Abmart, Shanghai, China) and GAPDH (1:5000, ab181602, Abcam) overnight at 4℃. After incubated with Goat Anti-Rabbit IgG second antibody (1:5000, M21003, Abmart), the membranes were visualized by Odyssey infrared imaging system (Tanon, Shanghai, China).

Xu M.M., Kang J.Y., Wang Q.Y., Zuo X., Tan Y.Y., Wei Y.Y., Zhang D.W., Zhang L., Wu H.M, & Fei G.H. (2024). Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis. Respiratory Research, 25, 186.

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MUC5AC Expression Regulation in Cells

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A Lyn-specific siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies for histology were purchased from Santa Cruz Biotechnology: MUC5AC (mucin 5AC, Clone # K-20; Cat # sc-16903), STAT6 (Clone # S-20, Cat # sc-621), β-actin (Clone # N-21, Cat # sc-130656), and phospho-STAT6 (Clone # Tyr641, Cat # sc-11762). The ELISA kits for IL-4, and IL-13 were purchased from Beijing Yonghui biotechnology Co. Ltd (Beijing, China). The MUC5AC-pGL3 luciferase vector was constructed by our laboratory based on the pGL3 luciferase reporter vector (Promega) following the manufacturer’s instructions.

Wang X., Li Y., Luo D., Wang X., Zhang Y., Liu Z., Zhong N., Wu M, & Li G. (2017). Lyn regulates mucus secretion and MUC5AC via the STAT6 signaling pathway during allergic airway inflammation. Scientific Reports, 7, 42675.

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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 Protein Expression

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Equal amounts of nuclear or whole cell extracts were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto a polyvinylidene difluoride membrane (Millipore, Schwalbach, Germany). The transformed membrane was blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for one hour and incubated with primary antibodies overnight at 4°C. The primary antibodies used were as follows: rabbit anti-arginase-1 (1:500), Ym1 (1:1,000), PPARγ (1:500), phospho-Stat6 (1:500, Santa Cruz, Heidelberg, Germany), β-actin (1:1,000, Santa Cruz, Heidelberg, Germany), lamin B1 (1:1,000, Santa Cruz, Heidelberg, Germany). The membrane was washed three times with TBST for ten minutes and incubated with anti-rabbit IgG-horseradish peroxidase (1:5,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for one hour. The Supersignal West Pico chemiluminescent substrate system (Millipore, Schwalbach, Germany) was used to detect immunoreactive bands. The intensity of protein bands after Western blot were quantitated by using Quantity One Version 4.6.3 Image software (Bio-Rad, Hercules, CA, USA) and normalized against proper loading controls.

Li L., Wu Y., Wang Y., Wu J., Song L., Xian W., Yuan S., Pei L, & Shang Y. (2014). Resolvin D1 promotes the interleukin-4-induced alternative activation in BV-2 microglial cells. Journal of Neuroinflammation, 11, 72.

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Fluorescent Conjugation of Food Antigens

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Food antigens [cow’s milk (Organic Valley Family of farms), and egg (Jay Robb Enterprises Inc) and ovalbumin) were conjugated to Alexa Fluor 647 dye as described by the manufacturer (ThermoFisher Scientific). The model antigens and inhibitors used were as follows: dextran, tetramethylrhodamine, 10,000 MW, lysine fixable and diamidino-2-phenylindole (DAPI) (ThermoFisher Scientific); 8-Br cADPR (Sigma); Tropicamide (SantaCruz); LY294002 (Cell signaling). The reagents used are as follows: Fluo4 AM and Imject Alum Adjuvant (ThermoFisher Scientific); RU486, polybrene, tamoxifen, carbachol, histamine and ovalbumin (Sigma Aldrich), human and mouse IL-13 (PeproTech), and mouse anti-IgE antibody, clone EM-95 (provided by Fred D. Finkelman at CCHMC). Antibodies that we used are as follows: Chromogranin A (Immunostar), MMP7 (R&D systems), mouse MCPT-1 (eBioscience), MUC2, STAT6, IL-4Rα, phospho-STAT6 (SantaCruz), phospho-AKT (Ser473) and phospho-AKT (Thr308) (#9275, Cell Signaling), actin (A2066, Sigma), DCLK1 and anti-GFP (Abcam), PerCP-Cy5.5 conjugated CD45R/B220, CD8, Ly-6G/Ly-6C, CD11c, and CD3e, PE/Cy7 conjugated ckit, biotin conjugated ST2 (BioLegend), streptavidin conjugated APC-Cy7 (BD Pharmigen), and APC conjugated FcϵR (BioLegend), Donkey anti-mouse, -rat, -rabbit & -goat conjugated to Alexa Fluor-488 or −647 (Invitrogen).

Noah T.K., Knoop K.A., McDonald K.G., Gustafsson J.K., Waggoner L., Vanoni S., Batie M., Arora K., Naren A., Wang Y.H., Lukacs N.W., Munitz A., Helmrath M., Mahe M.M., Newberry R, & Hogan S.P. (2019). IL-13-induced Intestinal secretory epithelial cell antigen passages are required for IgE-mediated food-induced anaphylaxis. The Journal of allergy and clinical immunology, 144(4), 1058-1073.e3.

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