Immunoblots were prepared using Bolt Bis-Tris gel systems (Invitrogen) and probed overnight at 4°C with antibodies to NOD1 (3545, Cell Signaling), IκBα (4814, Cell Signaling), p-p38 (4511, Cell Signaling), p-AKT (4060, Cell Signaling), pan-AKT (4691, Cell Signaling), C-MYC (13987, Cell Signaling), C-KIT (Ab 81, Santa Cruz), ALX1 (ab181101, abcam) or ERK2 (C-14, Santa Cruz). Anti-FLAG antibody conjugated to HRP (A8592, Sigma) was probed at room temperature for 30 min. Blots were visualized using SuperSignal West Dura chemiluminescent substrate (ThermoFisher) at a high or low exposure as indicated. For native gel analysis of NOD1 oligomers, cells were lysed using lysis buffer containing 1% NP-40. Cell debris were separated by centrifugation at 11000g for 15 min. Native sample buffer was added to the cell lysate and run on a 3–12% native Bis-Tris gel as per the manufacturer’s instructions (ThermoFisher). Proteins were transferred to a nitrocellulose membrane followed by immunoblotting.
Ab181101
Manufactured by Abcam
Ab181101 is a lab equipment product manufactured by Abcam. It is a core component designed for use in various laboratory applications. The product's primary function is to facilitate specific experimental procedures, but a detailed description is not available at this time while maintaining an unbiased and factual approach.
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Lab products found in correlation
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
A8592, by Merck Group (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Precast gel, by Thermo Fisher Scientific (1 mentions)
Pierce supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Mammalian protease inhibitor cocktail, by Merck Group (1 mentions)
Vin 11 5, by Merck Group (1 mentions)
2 protocols using ab181101
1
Immunoblot Analysis of NOD1 Signaling
2
Immunoblotting for Protein Expression
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Cells were lysed in radioimmunoprecipitation assay buffer containing mammalian protease inhibitor cocktail (Sigma). Equal protein amounts were separated by gel electrophoresis on precast gels (Life Technologies), transferred to nitrocellulose, and immunoblotted with antibodies to PLAC8 (ab122652, Abcam, Cambridge, MA, and HPA040465, Sigma), ALX1 (ab181101, Abcam), NOS3 (610296, BD Biosciences, San Jose, CA), or vinculin (VIN11-5, Sigma). Secondary antibodies conjugated to horseradish peroxidase were from Biorad (Hercules, CA). Blots were developed with Pierce Supersignal West Pico (ThermoFisher, Hanover Park, IL), exposed to film, scanned, and compiled in Photoshop CS5.1 (Adobe, San Jose, CA).
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