Ptal luc

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.

The putative promoter (nucleotides −1436 to +118 relative to the TSS) of PA28γ was amplified from human genomic DNA and inserted into the promoterless firefly luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA), and the resulting construct was denoted p(−1436/+118)-luc. Subsequently, a series of promoter deletion constructs was generated by PCR using p(−1436/+118)-luc as a template. A fragment of the PA28γ promoter (nucleotides −975 to −407) containing three putative p53 response elements (REs) was inserted upstream of the minimal TATA-like promoter in the firefly luciferase reporter vector pTAL-Luc (Clontech, Mountain View, CA, USA), and the resulting construct was denoted pwtREs-Luc. Site-directed mutagenesis of the construct pwtREs-Luc was performed by overlapping PCR. To generate the PA28γ expression construct pHA-PA28γ, the coding region of human PA28γ was cloned in-frame with Myc-tag into pCMV-HA. The HA-tagged p53 expression plasmid pHA-p53 was described previously [27 (link)].