( to 550) of NF- k B p65 was ...)New Mammalian Two-Hybrid System Detects Protein-Protein Interactions

Detect Interaction Between p53 and SV40 Large T-Antigen

Fig.4

The ability of the mammalian two-hybrid assay kit to detect protein-protein interactions was verified by the expression of p53 and SV40 large T-antigen. Amino acids 72 to 390 of murine p53 were fused in frame to the carboxyl (C) terminus of the GAL4-DNA binding domain (pBD-53), and amino acids 84 and 708 of the SV40 large T-antigen were fused to the C-terminus of NF-kB p65 activation domain (pAD-SV40T). As negative controls, amino acids 218 to 277 of CD40, ¹³ which does not interact with SV40T, were fused to the C-terminus of GAL4 DNA binding domain (pBD-CD40), and amino acids 297 to 503 of TRAF-2,¹³ which does not interact with p53, were fused to the C-terminus of NF-kB p65 activation domain (pAD-TRAF). These pairs of plasmids were cotransfected with the reporter plasmid pFR-Luc into various cell lines, and their luciferase activities were monitored. The cell lines used were CHO, COS, HeLa, 293, and HLR (HeLa luciferase reporter). Figure 4 shows data for luciferase expression in the various cell lines; transfection with the combination of plasmid pBD-53 and pAD-SV40T, whose expressed fusion proteins interact in vivo, resulted in strong activation of luciferase gene expression. Conversely, transfections with the combination of plasmids pBD-CD40 and pAD-SV40T or pBD-53 and pAD-TRAF2, whose expressed fusion proteins do not interact in vivo, displayed low background luciferase activity. Only combinations of pBD-53 and pAD-SV40T dramatically activated
'"/>

Source: