Figure 1.

N&B approach to discern dimerization status of RelA. (A) There are five homologous proteins of the NF-κB TF family. All contain a Rel homology domain (RHD) that promotes dimerization with other RHD-containing proteins as well as DNA binding. RelA, RelB, and c-Rel each also contain a transcription-activation domain (TAD) that enables them to activate transcription. Cleavage of p105 and p100 produces p50 and p52, respectively. Additional domains include: LZ, leucine zipper; GRR, glycine-rich region; ANK, ankyrin-repeat domain; DD, death domain. (B) 15 potential NF-κB dimers exist based on RHD interactions. (C) The N&B assay measures the oligomer state of a protein by determining its molecular brightness (ε) within a region of interest (ROI) in a cell. A fluorescent protein's molecular brightness is determined by calculating the fluctuations (variance) in mean fluorescence intensity (<I>) that are caused by the movement of protein oligomers (monomers, dimers, trimers, k-mers) within every pixel (confocal volume) of the ROI over time (confocal imaging acquisition). The ratio of the variance to the mean fluorescence intensity of the pixels is equal to the protein's brightness (ε) + 1. Because immobile proteins do not produce such movement-based fluctuations, their molecular brightness is equal to 0. (D) For quantifying a protein's brightness within an ROI, in our case the nucleus, the brightness values of each pixel comprising the ROI are extracted from a stack of N&B images (see Methods). The brightness values are then fitted to a Gaussian distribution to determine the protein's overall brightness within the ROI.