Total liver protein was extracted from mice liver using RIPA buffer (Boston Bio-products cat # BP-115) supplemented with protease inhibitor (Roche Cat. # 11836153001) and phosphor-Stop (Roche cat # 04906837001). 50ug of total protein was used for western blot analysis and resolved proteins transferred unto nitrocellulose membranes and visualized by chemiluminescent methods using the Fujifilm LAS-4000 luminescent image analyzer. The following antibodies were used: SYK (cell Signaling cat. # 2712), phospho-SYKY525/526 (Cell signaling cat. # 2710), phospho-SYKY525/526 (Abcam Cat. # ab58575), Phospho-NF-κB p65 (Ser536) (Cell signaling Cat. # 3031 and β-Actin (Abcam cat. # ab6276).
Phospho syky525 526
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-SYKY525/526 is a monoclonal antibody that recognizes the phosphorylated forms of the SYK (spleen tyrosine kinase) protein at the Tyr525 and Tyr526 residues. This antibody can be used to detect the activated, phosphorylated state of SYK in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Phospho nf κb p65 ser536, by Cell Signaling Technology (2 mentions)
Phosphor stop, by Roche (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
β actin, by Abcam (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Nlrp3, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bca method, by Solarbio (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Western lightning ecl, by PerkinElmer (1 mentions)
Phosstop phosphatase inhibitor, by Roche (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 protocols using phospho syky525 526
1
Quantification of Liver Protein Signaling
2
Lung Protein Expression Analysis
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The lung tissues or cells cultured in vitro were lysed and centrifugated at 12,000 ×g for 10min at 4 °C, and then the supernatants were removed and quantified via the BCA method (Solarbio, Beijing, China). Equal amounts of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto polyvinylidene difluoride membranes (PVDF, Millipore, Bedford, MA, USA) and blocked with 5% skim milk for 1 h at room temperature. The membranes were first probed with specific primary antibodies against Syk (#13198S), phospho-Syk (Y525/526) (#2710S), NF-κB p65 (#8242S), phospho-NF-κB p65 (Ser536) (#3033S), NLRP3 (#15101S), iNOS (#13120S) and β-actin (#4970) (Cell Signaling Technology, Boston, MA, USA) separately overnight at 4 °C. After washing, they were incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibodies for 1 h at room temperature and then visualized with an enhanced chemiluminescence kit (ECL, Thermo Scientific, Rockford, IL, USA) on a Chemical imaging system (Clinx Science Instruments Co., Ltd, Shanghai, China). The relative intensities of bands were quantified using Image-J software (National Institutes of Health, Bethesda, Maryland, USA).
3
Western Blot Analysis of SYK Phosphorylation
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Whole-cell lysates were extracted in 1 × Cell Lysis Buffer (Cell Signaling, Boston, MA, USA) supplemented with EDTA-free protease inhibitors and PhosSTOP phosphatase inhibitors (Roche). Western immunoblotting was performed as follows. Briefly, 10–50 μg of protein was loaded on an SDS-PAGE gel and transferred to a 0.45 μM nitrocellulose membrane. Membranes were blocked in 5% bovine serum albumin (BSA; Sigma-Aldrich) in Tris-buffered saline with 1% Tween-20 (TBST), incubated in primary antibody overnight at 4°C, incubated in secondary antibody for 2 hours at room temperature, and developed using Western Lightning ECL (Perkin Elmer, Waltham, MA, USA). Primary antibodies against total SYK and Actin were purchased from Santa Cruz Biotechnology. The phospho-SYK (Y323) primary antibody was obtained from Epitomics (now Abcam, Cambridge, MA, USA) and the phospho-SYK (Y525/526) from Cell Signaling (Boston, MA, USA).
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