Pp53 ta luc

The reporter vector pp53-TA-Luc (Clontech Carlsbad, CA, USA) was used to quantify TP53 transcriptional activity. The vector contains a TP53 responsive element located upstream the TATA box from the herpes simplex virus timidine kinase promoter (pTA). Downstream of pTA is the firefly luciferase reporter gene (Luc). The pRL-TK vector contains the herpes simplex virus thymidine kinase promoter to provide low to moderate levels of Renilla luciferase expression in co-transfected mammalian cells. The vector was used as an internal control reporter in combination with pp53-TA-Luc reporter vector. Luciferase activity was measured using a dual luciferase kit (Promega, WI, USA) and quantified at a luminometer (Turner, Biosystems, Sunnyvale, CA, USA). Each condition was assayed in four replicates in two independent experiments. The TP53 mammalian expression vectors pc53SN, which carries a human wild type TP53 cDNA were a kind gift of Dr. Arnold Levine and were previously described [45 (link)].

The genes HPV6 E6, HPV6 E7, HPV11 E6, HPV11 E7, HPV16 E6 and HPV16 E7 were amplified by PCR containing restriction enzyme sites. These PCR products were digested with restriction enzymes, Bgl II and Xho I (New England Biolab), and ligated to pMSCV-puro vector (Clontech). Plasmids expressing EBV LMP-1, (i.e. pMSCV-neo-LMP-1) were prepared as previously described [15 (link)]. pNF-κB-TA-luc, pp53-TA-luc, pβ-gal-basic (Clontech) and pcDNA3.1/Zeo (+) (Invitrogen) were also used for assays.

The plasmid expressing DNM IκB-α, the pSV-β-galactosidase control vector, and the reporter plasmids of pNF-κB-Luc and pp53-TA-Luc were purchased from Clontech (Palo Alto, CA), Promega Corp. (Madison, WI), and Stratagene Inc. (La Jolla, CA), respectively. The plasmid or control vector was transfected into cells by use of Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions.