Renilla luciferase substrate

U373 cells were cotransfected with a firefly luciferase reporter plasmid and the internal control CMV Renilla luciferase plasmid. Twenty-four hours later, cells were treated with increasing concentrations of FTY-P or vehicle control. One hour later, TNF-α was added as indicated, and 8 h later, cells were lysed with passive lysis buffer (Promega, Mannheim, Germany). Reporter gene activity was determined using firefly luciferase substrate (Biozym, Hamburg, Germany) and Renilla luciferase substrate (Promega), respectively.

To measure NF-κB activation, HEK293T cells were transiently transfected with a plasmid containing a firefly luciferase reporter gene under the control of an NF-κB transcriptional response element, a plasmid with a Renilla reniformis luciferase reporter gene for normalization and the indicated amounts of expression or control plasmids using Lipofectamine 2000 (Invitrogen Life Technologies). We used human BCMA or human TACI for transfection. To determine the effect of γ-secretase inhibition on NF-κB activation, cells transfected with BCMA were treated with DAPT or a solvent control and 6 h later stimulated with APRIL or BAFF. To analyse a possible decoy function, DAPT along with BAFF or APRIL were added to BCMA-Fc or supernatants generated by HEK293T cells that had been transfected with full-length BCMA or an empty control vector as described above. These supernatants were incubated at 37 °C for 30 min and then added to BCMA or TACI-transfected and DAPT-treated cells used for the reporter assay. 16 h after stimulation cells were harvested and cell lysates were prepared using passive lysis buffer (Promega, Madison, WI, USA) and the reporter gene activity was measured using firefly luciferase substrate (Biozym, Hameln, Germany) and renilla luciferase substrate (Promega) respectively.