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The transcription factor NFκB (nuclear factor κB) is a key component for the inducible expression of a wide variety of cellular and viral genes. NFκB is composed of a heterodimer of p65 and p50 subunits in most cell types and is sequestered in the cytoplasm by its inhibitory proteins, the IκBs. During the phosphorylation and degradation of IκBs, NFκB p65 is phosphorylated on multiple resides, each triggered by different stimuli but essential for maintaining NFκB transcriptional activation. Because of its role in regulating inflammatory and immune responses, high-throughput study methods for monitoring NFκB phosphorylation are in high demand.
Figure 1: Measurement of phosphorylated and total NFκB p65.
HeLa cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 20 ng/ml TNF-α and 50 nM Calyculin A for 15 minutes and fixed. Total and phospho NFκB p65 were each assayed in triplicate using the phospho and total NFκB p65 antibodies included in the FACE NFκB p65 Profiler Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).
Antibody Specificities
The phospho-NFκB p65 (S468) was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 468 of human NFκB p65 and does not cross-react with other sites. The phospho-NFκB p65 (S536) was raised against a synthetic phospho-peptide corresponding to residues surrounding Serine 536 of human NFκB p65 and does not cross-react with other sites. The total-NFκB p65 antibody recognizes NFκB p65 proteins regardless of the phosphorylation state.
