NF-kB is a family of transcription factors, which regulates thousands of genes expression and plays an important role in various cellular response. NF-kB function as dimers. In resting cells, the p52 homodimer, in the absence of its binding partner Bcl3, acts only as a repressor of transcription since it does not have TAD. However, the p52 homodimer gain the transcriptional regulation ability by binding to atypical IkB protein Bcl3. Bcl3 is a super-phosphorylated protein in nature. Un-phosphorylated Bcl3 is not competent on directing transcription activation. Three phosphorylation sites have been identified to be critical for Bcl3’s cellular activities. Akt phosphorylates Ser33 of Bcl3 which is required for its nuclear translocation and stabilization of the protein; Erk2 phosphorylates Ser114 and IKK1/2 phosphorylates Ser446, this two latter phosphorylation are essential for Bcl3’s transcriptional regulation. Recently, we identified the fourth phosphorylation site of Bcl3. Phosphorylation on Ser446 can largely enhance the Bcl3’s ability of transcriptional activation. Here, to understand the functional relationship of p52, Bcl3 and DNA, we purified p52 and phospho-mimic Bcl3(4E) proteins in vitro and performed Isothermal titration calorimetry (ITC) assay to study their binding affinity.