Heart tissue protein was extracted in RIPA lysis buffer (Pierce) supplemented with protease/phosphatase inhibitor cocktail (Cell Signaling). Total protein concentration was quantified using BCA assay (Pierce). Lysates were denatured and subjected to gel electrophoresis using NuPAGE Novex Gel system (Life Technologies) and blotted on nitrocellulose membrane using iBlot Gel Transfer system (Life Technologies) according to the manufacturer’s instructions. Blotting was performed with rabbit anti-mouse total IRF3 (Cell Signaling, #4302, Clone D83B9, used at 1:1000 dilution) and Phospho-IRF3 (Ser396) (Cell Signaling, #4947, Clone 4D4G, used at 1:1000 dilution). Loading control was blotted with goat anti-mouse GAPDH antibody (R&D Systems, P04406, used at 1:1000 dilution). Blots were visualized by labeling with HRP-coupled anti-rabbit and anti-goat secondary antibodies (Pierce, used at 1:5000) and incubating with chemiluminescent substrate (Pierce) administered. Gels were developed on a Kodak automated developer and quantified using densitometry with ImageJ. Quantification of CXCL10 protein was performed using an ELISA kit (R&D Systems, MCX100) according to manufacturer recommendations.
Clone d83b9
Manufactured by Cell Signaling Technology
Clone D83B9 is a monoclonal antibody that specifically targets the IL-6 receptor. It can be used for detection and analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Iblot gel transfer system, by Thermo Fisher Scientific (2 mentions)
Novex nupage gel system, by Thermo Fisher Scientific (2 mentions)
Clone 4d4g, by Cell Signaling Technology (2 mentions)
Goat anti mouse gapdh antibody, by R&D Systems (2 mentions)
Mcx100, by R&D Systems (2 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (2 mentions)
2 protocols using clone d83b9
1
Immunoblot Analysis of IRF3 Activation
2
Immunoblot Analysis of IRF3 Activation
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Heart tissue protein was extracted in RIPA lysis buffer (Pierce) supplemented with protease/phosphatase inhibitor cocktail (Cell Signaling). Total protein concentration was quantified using BCA assay (Pierce). Lysates were denatured and subjected to gel electrophoresis using NuPAGE Novex Gel system (Life Technologies) and blotted on nitrocellulose membrane using iBlot Gel Transfer system (Life Technologies) according to the manufacturer’s instructions. Blotting was performed with rabbit anti-mouse total IRF3 (Cell Signaling, #4302, Clone D83B9, used at 1:1000 dilution) and Phospho-IRF3 (Ser396) (Cell Signaling, #4947, Clone 4D4G, used at 1:1000 dilution). Loading control was blotted with goat anti-mouse GAPDH antibody (R&D Systems, P04406, used at 1:1000 dilution). Blots were visualized by labeling with HRP-coupled anti-rabbit and anti-goat secondary antibodies (Pierce, used at 1:5000) and incubating with chemiluminescent substrate (Pierce) administered. Gels were developed on a Kodak automated developer and quantified using densitometry with ImageJ. Quantification of CXCL10 protein was performed using an ELISA kit (R&D Systems, MCX100) according to manufacturer recommendations.
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