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to 550) of NF-kB p65 was used as the activation domain in pAD.
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
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