Western blot analysis was performed as previously described8 (link). In brief, total protein samples extracted from untreated and treated tissues or cells were prepared using RIPA buffer containing a protease inhibitor cocktail (Roche, Branford, CT, USA) and a phosphatase inhibitor and were subjected to SDS PAGE (90μg protein per sample). The membranes were then incubated with the following primary antibodies: p-IRS-1 (Cell Signaling Inc. (CST), Danvers, MA, USA,1:800), eNOS (CST,1:1,000), p-eNOS (Thermo Fisher,1:800), IRS-1 (CST,1:1,000), p-AKT (CST,1:1000), AKT (CST,1:1000), p-PI3K (Abcam, Cambridge, MA, USA,1:500), PI3K(Abcam1:1000),GAPDH (CST, 1:1,500), anti-LC3 (1:200, Sigma-Aldrich, St. Louis, MO, USA), Beclin1 (1:200, Sigma-Aldrich), GLUT4 (1:3,000, Thermo Fisher), p-JAK2 and JAK2(CST,1:1000).
P jak2
Manufactured by Thermo Fisher Scientific
Sourced in United States
P-JAK2 is a lab equipment product designed for the detection and analysis of phosphorylated Janus Kinase 2 (p-JAK2) protein. It is a tool used in research and clinical applications to study cell signaling pathways involving the JAK2 protein.
Automatically generated - may contain errors
Lab products found in correlation
P stat3, by Thermo Fisher Scientific (2 mentions)
P irs1, by Cell Signaling Technology (1 mentions)
P akt, by Abcam (1 mentions)
Irs 1, by Abcam (1 mentions)
P enos, by Thermo Fisher Scientific (1 mentions)
Glut4, by Thermo Fisher Scientific (1 mentions)
Anti lc3, by Merck Group (1 mentions)
Beclin 1, by Merck Group (1 mentions)
Gapdh, by Merck Group (1 mentions)
P pi3k, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Complete freund s adjuvant cfa, by Merck Group (1 mentions)
Tadalafil, by Eli Lilly (1 mentions)
Ab97030, by Abcam (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Pd l1, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sh ptp1, by Santa Cruz Biotechnology (1 mentions)
Socs3, by Thermo Fisher Scientific (1 mentions)
Ptyr705 stat3, by Cell Signaling Technology (1 mentions)
5 protocols using p jak2
1
Western Blot Analysis of Protein Signaling
2
Comprehensive Pharmacological Evaluation of Methotrexate and Anti-Inflammatory Agents
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Methotrexate and prednisolone were obtained from (Mylan, France) and Egyptian Pharmaceutical Industries Co. (EIPICO, Egypt), respectively. The sources of tadalafil and aliskiren were Eli Lilly & Co. and Novartis Pharmaceuticals Corporation, respectively. Cinnamaldehyde (purity ≥ 98%) was purchased from LOBA Chemie (Mumbai, India) for laboratory reagents and fine chemicals. Complete Freund's adjuvant (CFA) was purchased from Sigma-Aldrich Co. (USA). The sources of RF, TNF-α, IL-6, IL-10, MMP-3, RANKL, and myeloperoxidase (MPO) ELISA kits were CUSABIO (Bio-Connect Diagnostics, The Netherlands) and MyBioSource (USA). The colorimetric kits of malondialdehyde (MDA), reduced glutathione (GSH), and nitric oxide (NO) measured as nitrite were purchased from Bio-Diagnostic Co. (Egypt). The primary antibodies for Western blot analysis, including p-JAK2, p-STAT3, iNOS, and eNOS were obtained from ThermoFisher Scientific (USA).
3
Immunofluorescence Staining of JAK2, Src, and STAT3
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The cell layers were fixed with 3.7% paraformaldehyde at room temperature for 20 min and then permeabilized by washing five times with 0.1% Triton X-100 for 5 min each time. Subsequently, the cell monolayers were blocked with 5% BSA for 90 min and then incubated with antibodies against JAK2 (1:50, PA5-11267, ThermoFisher), p-JAK2 (1:500, PA5-105889, ThermoFisher), Src (1:50, MA5-11173, ThermoFisher), p-Src (1 μg/mL, 44–662G, ThermoFisher), STAT3, or p-STAT3 for overnight at 4°C. After washing with 0.2% PBST, the cell layers were stained with goat anti-rabbit IgG or goat anti-mouse IgG, washed, imaged with a two-color infrared laser imaging system, and analyzed using the Image Studio software.
4
Western Blot Analysis of Tumor Cells
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Tumor cells were collected following centrifugation at 300 × g for 5 min at 4°C) and supernatant removal. Cells were lysed in RIPA buffer (50 mM Tris-Cl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.2 mM PMSF). Proteins (20–40 µg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (EMD Millipore). The PVDF membrane was placed in blocking buffer and put on shaker for 30 min at room temperature and wash three times with PBS for 10 min. Subsequently, membranes were incubated with primary antibodies (1:1,000), for 1 h at room temperature on a shaker. Following 3 washes for 10 min each, with PBS with Tween 20, HRP-conjugated secondary antibodies (1:5,000; ab97030; Abcam) incubated for 1 h at room temperature on a shaker. Finally, band intensities were visualized in Imager (Bio-Rad Laboratories) using ECL system (34095; Thermo Fisher Scientific, Inc.). Primary antibodies used in the assay included: Phosphorylated (p)-ATM (MA1-46069; Thermo Fisher Scientific, Inc.), PD-L1, p-JAK1, p-JAK2 and p-STAT3 and GAPDH (14-5983-82, 44–422G, 710928, 44–384G, MA5-15738; Thermo Fisher Scientific, Inc.).
5
Immunoblotting of Hepatic Signaling
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Western blots for immunocomplexes and hepatic homogenates were performed using antibodies against phosphorylated (p) Tyr705-signal transducer and activator of transcription factor 3 (pTyr705-STAT3), pSer727-STAT3 and SOCS3 from Cell Signaling Technology (Danvers, MA), pTyr1007/1008 Janus kinase 2 (pJAK2), JAK2 and regulatory subunit of PI3K (p85) from Millipore (Temecula, CA), STAT3 from R&D Systems (Minneapolis, MN) and aquaglyceroporin-9 (AQP9), the beta chain of insulin receptor (IRb), catalytic subunit of PI3K (p110), GLUT2, long form of the leptin receptor (OBeRb), phosphoenolpyruvate carboxykinase (PEPCK) and SH-PTP1 from Santa Cruz Biotechnology (Santa Cruz, CA). The proteins were detected by chemiluminiscence using an ECL system. Quantification of the bands was carried out by densitometry using a Kodak Gel Logic 1500 Image Analysis system and Molecular Imaging software 4.0 (Rochester, NY, USA). AQP9, GLUT2, IRb, OB-Rb, p85, PEPCK, SOCS3 and SH-PTP1 were normalized with actin (Thermo Scientific, Fremont, CA), whereas pJAK2, pTyr705-STAT3 and p-Ser727-STAT3 were normalized with their respective total forms.
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