Prl tk renilla luciferase

C2C12BRA cells were stably transfected with a BMP-responsive, Id1 promoter, firefly luciferase reporter gene construct to assess BMP signaling, as previously described^{54 (link),55 (link)}. Lipofectamine (Invitrogen) was used to transfect cells containing the reporter gene with wild-type ALK6, rs34970181 (R371Q) ALK6, or constitutively active (Q204D) ALK6 (kindly provided by Dr. Takeshi Imamura, JFCR Cancer Institute, Tokyo, Japan). Transfection efficiency was determined using pRL-TK renilla luciferase (Promega). The ratio of firefly to renilla luciferase activities was calculated to quantify the amount of BMP signaling normalized to transfection efficiency. For BMP stimulation experiments, transfected C2C12BRA cells were incubated for 16 hours in serum-free media together with 0, 5, or 25 ng/ml of BMP2.

To test Gli promoter activity, reporter assay was performed with Gli-responsive luciferase reporter construct (8×Gli-Luc) (gift from Dr. Fernandez-Zapico^{48 (link)}). DPSCs (1×10⁶) were transfected with 3 μg 8×Gli-Luc and 0.6 µg pRL-TK Renilla luciferase (Promega, internal control) with Fugene HD transfection reagent. Cells were induced with OS medium for 3 days and then stimulated with 1 μg/mL of recombinant mouse Shh N-terminus (R&D Systems, Minneapolis, MN, USA) for 8 h. Cells were harvested and colored by the Dual-luciferase Assay Kit (Promega, Madison, WI, USA) and the relative luciferase activity was measured with the Veritas microplate luminometer (Turner Biosystem, Sunnyvale, CA, USA). The experiment was conducted in triplicate.

COS-7 cells (purchased from ATCC) were maintained at 37°C and 5% CO₂ in DMEM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM GlutaMax. On the day of the transfection the cells were 75% confluent. Cells were transfected by Neon electroporation according to the manufacturers instructions (Invitrogen). Briefly, 500 ng of each protein expression construct, IL2-firefly-Luc reporter [14] (link), and pRL-TK-Renilla-luciferase (Promega) were mixed with the either aptamer-19 or its antisense sequence (AS) as a control; the oligos contained phosphorothioate linkages at the three terminal positions on both ends to increase cellular stability. Scrambled DNA (oligo of the same chemical composition as aptamer-19, but with a different sequence; 5′-GTGACACGAATTGGGACCAGCGTATGGCTGATATAACATGTTTCGACCGAGCCTGACCGGTTG-3′) was used to maintain an equal amount of oligonucleotide DNA for each reaction. Cells were electroporated and seeded into individual wells of a 6-well plate in the absence of antibiotics. 16 hr post transfection, cells were stimulated with 1 µM ionomycin and 20 ng/ml PMA for 6 hr. Cells were then harvested and lysed with 250 µl Passive Lysis Buffer (Promega). Firefly and renilla luciferase activities were determined using the Dual-luciferase kit (Promega).

Plasmids pGL3-ARE-luc were used to analyze the NRF2 activity. pGL4-NF-κB-RE-luc (Promega, USA) was used to analyze the NF-κB activity. The pRL-TK Renilla luciferase (Promega, USA) plasmid was used to control for transfection efficiency. The activities of Firefy and Renilla luciferase were determined using the dual-luciferase reporter assay system (Promega, USA) according to manufacturer instructions. Assays were independently conducted at least in triplicate. The data presented show relative Firefy luciferase activity normalized to Renilla luciferase activity.

Stable cell lines (CMV-TRAF6 cells or TRAF6-sh2 cells) in 24-well plates were transfected with 500 ng pNF-κB-luc and 50 ng pRL-TK Renilla luciferase (Promega, USA), the pRL-TK plasmid as a control for transfection efficiency. At 12 hpt, cells were mock-infected, CSFV-infected, or ligand-stimulated for an additional 12 h. The activities of Firefly and Renilla luciferase were determined using the dual-luciferase reporter assay system (Promega) according to manufacturer instructions. Assays were conducted the independent experiments at least in triplicate. The data represent relative Firefly luciferase activity normalized to Renilla luciferase activity.