Pnf kb luc

For the construction of the luciferase plasmids, four copies of each kappaB site or mutant site (see below) were ligated (LigaFast rapid DNA ligation System, Promega, WI, USA) into a BglII and MluI-digested pGL2-Basic vector (Promega). The insert was generated by annealing a synthetic 5' phosphorylated oligonucleotides (Syntezza, Jerusalem, Israel). A control plasmid containing the consensus NF-kappaB sequence linked to the luciferase sequence was obtained from Clontech (pNF-kB-Luc, CA, USA). The CMVp65 and CMVdeltaNIkappaB plasmids have been previously described [35] .
The sequences of the kappaB sites inserted into the luciferase vector are as follows:
MGMT-kB1-Luc: 4XGTAAAGTCCCC
MGMT-Mut-kB1-Luc: 4XGTAAAGTCGGC
MGMT-kB2-Luc: 4XGGAACACCCC
MGMT-Mut-kB2-Luc: 4XGTAAAGTCGGC

HEK293T cells seeded in 24-well plates at 4 × 10⁵ cells/ml were transfected with a suitable amount of vectors expressing TNFR1 or TNFR1-d2 fused to Flag tag (from 10 to 70 ng) along with 10 ng β-galactosidase vector and 50 ng of a vector containing luciferase firefly cDNA under the control of a constitutive promoter region combined or not with four copies of the NF-kB consensus sequence (pNFkB-luc or pTAL-luc, respectively, Clontech). Total amount of transfected vector (800 ng) was obtained by the addition of an appropriate amount of empty pcDNA vector. After 24 h, cells were harvested and β-galactosidase activity was measured in 20 µl cleared lysates as described in the bicistronic reporter assay section. By using the Thermo Scientific Varioskan Flash Multimode Reader, firefly luciferase activity was measured on 50 µl cleared lysates diluted five times after a 50-µl single injection of buffer containing 530 µM ATP (Sigma Aldrich), 470 µM luciferin (Sigma Aldrich), and 270 µM coenzyme A (Sigma Aldrich) in luciferase buffer [40 mM Tricine, pH 7.8; 2.14 mM (MgCO₃); 4 Mg(OH)₂, 5H₂O; 5.34 mM MgSO₄; 0.2 mM EDTA]. The relative firefly luciferase activity for each condition was normalized to β-galactosidase activity and was subtracted by the basal luciferase activity obtained with the pTAL-luc vector.