Mammalian two hybrid system

HeLa cells (100 000) per well (six-well plate) were transfected 18–24 h after plating with 0.2 µg of luciferase expression plasmids [luciferase expression is driven by either CMV (pGL3 pCMV, Promega) or L1 5′UTR promoters (52 (link))], 0.02 µg of the MT1 expression plasmid and 0.02 µl of the pBIND plasmid (Mammalian two-hybrid system, Promega), which expresses Renilla luciferase. pGL3-basic (Promega) was used as a negative control. DNA was transfected using 1 µl of Plus and 1.5 µl of lipofectamine reagents (Invitrogen). The transfection cocktail was replaced with regular media 3 h after transfection. Cells were harvested for luciferase activity analysis 48 h after transfection. Protein extraction was performed according to the manufacturer's protocol (Promega). Protein concentrations were determined using standard BSA approach. The same amount of protein for each sample was analyzed using Promega dual-luciferase detection system.

Mammalian two-hybrid analyses were performed using a commercially available mammalian two-hybrid system (Promega, Madison, WI, USA), which includes the pACT, pBIND and pG5luc plasmids. The pG5luc reporter plasmid contains five GAL4-binding sites upstream of a minimal TATA box that precedes the firefly luciferase gene. The coding sequences of proteins were also generated by PCR, and cloned into pACT and pBIND at the BamHI/SalI sites. The nucleotide sequence of the inserts was verified by sequence analysis, and expression of all proteins was verified by Western blot analysis.
The luciferase assays were performed according to the manufacturer's recommendation. Approximately 48 h after each transfection, the luciferase activity was detected and normalized by Renilla activity.