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2 protocols using anti p jak2 antibody

1

Eriodictyol Inhibits Inflammatory Pathways

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Eriodictyol (EDT) (≥95.0%, Cat No. 89061), AG 490 (ak-2 protein tyrosine kinase inhibitor, Cat No. T3434) and 2,3,5-Triphenyltetrazolium chloride (TTC, ≥95.0%, Cat No. T8877), were purchased from Sigma-Aldrich (Shanghai, China). Lactate dehydrogenase (LDH) Cytotoxicity Assay Kit (Cat No. C0017), Rat IL-6 Enzyme-Linked ImmunoSorbent Assay (ELISA) Kit (Cat No. PI328), Rat TNF-α ELISA Kit (Cat No. PT516) and Colorimetric TUNEL Apoptosis Assay Kit (Cat No. C1098) were purchased from Beyotime Biotechnology (Shanghai, China). Creatine Kinase Activity Assay Kit (Cat No. BC1140) Rat CRP EasyTestTM ELISA kit (Cat No. 1030026) were purchased from Beijing Solarbio Science & Technology Co., Ltd., Rat IL-8 ELISA Kit (Cat No. BP-E30583) was purchased from Shanghai Langton Biotechnology Co., Ltd., Anti-Bax antibody (Cat No. sc-7480), Anti-Bcl-2 antibody (Cat No. sc-7382), Anti-Caspase-3 antibody (Cat No. sc-56053), Anti-JAK2 antibody (Cat No. sc-390539), Anti-STAT3 antibody (Cat No. sc-8019), Anti-p-STAT3 antibody (Cat No. sc-8059) and anti-β-actin antibody (Cat No. sc-47778) were all purchased from Santa Cruz Biotechnology. Anti-p-JAK2 antibody (Cat No. ab32101) was purchased from Abcam (Shanghai, China).
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2

Western Blot Analysis of Signaling Proteins

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Cells were harvested and lysed in PRO-PREP protein extraction solution (iNtRON Biotechnology). Cell lysates were scraped, and protein concentrations were measured with the Bradford assay. Equal amounts of protein were denatured in 5×protein sample buffer (ELPIS biotech, Daejeon, Korea), separated by SDS-PAGE on 12% polyacrylamide gel, and transferred to Immun-Blot®PVDF membranes (Bio-Rad, Quarry Bay, Hong Kong). The membranes were blocked with 3% bovine serum albumin, and probed with the following primary antibodies overnight at 4°C: anti-leptin antibody (Abcam), anti-p-JAK2 antibody (Abcam), anti-p-STAT3 antibody (Cell signaling, Danvers, Massachusetts, USA), anti-Notch1 antibody (bioworld, Dublin, Ohio, USA), anti-NICD antibody (Abcam), anti-Jagged1 antibody (Abcam), anti-survivin antibody (Enzo Life Sciences, Farmingdale, New York, USA) or β-actin polyclonal antibody (Abcam). After washing, the membranes were incubated with goat anti-rabbit IgG antibody (H+L), HRP conjugated (Bioss Antibodies, Woburn, Massachusetts, USA) for 1 hr at room temperature, blots were visualized using Chemiluminescent Sensitive Plus HRP Microwell and/or Membrane Substrate (SurModics, Eden Prairie, Minnesota, USA) and signals were measured with a Chemi-Doc® (Bio-Rad, Hercules, California, USA) [26 (link)].
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