Figure 2
From: Cloning-free regulated monitoring of reporter and gene expression
Dynamic response of the activity of reporter expressed from PCR product. (A) The reporter expression plasmid was used as a template for PCR in which the forward primers target either -53 or -95 sequences of the CMV promoter and includes two or four copies of NF-κB or a mutant NF-κB control (Table 1, SEQ. 1–3). (B) Fluorescence levels from EGFP PCR products that contain two, four, or mutant copies of NF-κB were quantitated as described above.(C) HEK293 cells were transfected with either NfκB-firefly luciferase expression vector or purified PCR products that were amplified from the luciferase vector and co-transfected with Renilla luciferase expression vector. Empty DNA represent either the vector or the PCR product without the inserted NF-κB sequence. The cells were treated with either IL-1α or TNF-α for 4 hrs as previously described. Luciferase activity levels were quantified by a luminometer and either absolute expression levels (C) or fold increase over control (no treatment) are shown (D). Data are Mean ± SEM. *p < 0.05, *** p < 0.001.