Transfections for reporter assays using RID deletion mutants in A293 and mouse 3T3 cells using Superfect and in DF1 cells using DMSO/polybrene were carried out as described previously (Kalaitzidis and Gilmore, 2002 (link); Starczynowski et al., 2007 (link)). Transfections using the Superfect reagent were performed according to the manufacturer's recommendations (Qiagen, Valencia, CA, USA). Transfections of A293 cells for EMSAs or reporter assays with REL splice variant isoforms were performed using PEI (polyethylenimine; Polysciences Inc., Warrington, PA, USA). A293 cells were seeded to be 40-60% confluent on the day of transfection. On the day of transfection, cells were incubated with DNA/PEI at a ratio of 3:1 in serum-free media (300 μl for a 60 mm plate) for 15min at room temperature. Following incubation, 4.7ml of DMEM/10% FBS was added to the DNA mixture, and this mixture was added to cells. Twenty-four hours later, the media was replaced with 5ml of fresh DMEM/10% FBS. The following day, the cells were harvested.
After lysing the cells, luciferase activity was measured using the Luciferase Assay System according to the manufacturer's recommendations (Promega, Madison, WI, USA). Luciferase values were normalized to βgal levels in all assays, as described previously (Kalaitzidis et al., 2002 (link)).
Yeast reporter gene assays were carried out as previously described (Epinat et al., 2000 (link)).
After lysing the cells, luciferase activity was measured using the Luciferase Assay System according to the manufacturer's recommendations (Promega, Madison, WI, USA). Luciferase values were normalized to βgal levels in all assays, as described previously (Kalaitzidis et al., 2002 (link)).
Yeast reporter gene assays were carried out as previously described (Epinat et al., 2000 (link)).
