Dual luciferase method

For the analysis of the MEF2 dependent luciferase activity, cells at 80–90% confluence were transiently transfected with plasmid pMEF2X4-E1b-Luc (a kindly gift of Prof. Brian Black, UCSF, USA) using LipofectAMINE2000 as described by the manufactured. The DNA mixture comprised the luciferase reporter and the reference plasmid pRL-TK (ratio 95:5).
Neuro2a cells were incubated in presence or absence of 25 μM HMB for 24 h. Luciferase activity was determined using the Dual Luciferase method (Promega) in a luminometer (Sirius L, Berthold Technologies, Bad Wildbad, Germany) and results were standardized for Renilla luciferase activity. To allow comparison of the expression patterns, the data are expressed as relative changes in luciferase activity and were normalized to a value of 100%.

For the analysis of rat Ubiquitin (UbC) promoter, a 417 bp fragment flanking the transcription start point was amplified from rat genomic DNA. This fragment is similar to those characterized for human and rat promoters [20 (link)]. Sequences of all constructs were verified by automated DNA sequencing. The Cignal FoxO Reporter (luc) kit contains a mixture of inducible FoxO-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).
For gene reporter analysis, cells were used at 80–90% confluence. Transfection was performed using LipofectAMINE2000 as described above. The DNA mixture comprised the pGL3-UbC luciferase reporter and the reference plasmid pRL-TK (ratio 95:5) or the Cignal FoxO Reporter mixture.
L6 myotubes were incubated in presence or absence of 25 μM HMB for 48 h. Cells were then incubated in the presence or absence of 5 μM DEX for 24 h. Luciferase activity was determined using the Dual Luciferase method (Promega) in a luminometer (Sirius L, Berthold Technologies, Bad Wildbad, Germany) and results were standardized for Renilla luciferase activity. To allow comparison of the expression patterns, the data are expressed as relative changes in luciferase activity and were normalized to a value of 100%.

Assays to measure activity of reconstituted RVFV RNPs were essentially performed as described (38 (link)). Briefly, subconfluent monolayers of 293 cells seeded in 12-well dishes were transfected with pI.18-RVFV_L, pI.18-RVFV_N (or mutants thereof), pI.18-HA-PKRΔE7 and pHH21-RVFV-vMRen (0.5 μg each), and the firefly luciferase control pGL3 (0.05 μg), using Nanofectin (PAA). At 24-h post-transfection, cell extracts were tested for Renilla and firefly luciferase activities using the Dual luciferase method (Promega).