Renilla luciferase reporter control plasmid

The LMNA (Rattus norvegicus (Norway rat), Gene ID: 60374) promoter region (P1 covers 1500 bp upstream of the transcription start site; P2 is from 3000 bp to 1500 bp upstream of the transcription start site) was cloned in to pmirGLO (Promega). Primary LSECs were co-transfected with 1 ug LMNA promoter luciferase plasmid and 100 ng Renilla luciferase reporter control plasmid (Promega). After transfection for 48 h, the luciferase activity of the above treated-LSECs and its renilla luciferase activity were measured with the Dual-Luciferase Reporter Assay System and normalized by renilla luciferase activity.

HT1080 cells or HeLa cells were plated in 96 well plates at 8000 cells per well and co-transfected, using the Lipofectamine LTX and Plus^TM reagent (Life technologies), with 80 ng of the various pCpGL plasmids and 20 ng of Renilla Luciferase Control Reporter plasmid (Promega) per well. The pCpGL plasmid containing no PLAT promoter sequence served as a negative control. In some experiments the protein kinase C activator phorbol 12-myristate 13-acetate (PMA)(Sigma-Aldrich) at a final concentration of 20 nM was added 24 hours after transfection. 36 hours after transfection, luciferase activity was measured using the Dual-Glo® Luciferase Reporter Assay System (Promega). Firefly luciferase activity of each individual transfection was normalized against Renilla luciferase activity. Each transfection was done in quadruplicate. Luminescence was measured using a Perkin Elmer Victor3 luminescence detection instrument (Perkin Elmer, Waltham, MA, USA).