SEAP assay

A NF-κB reporter cell line (293-SEAP) was used to monitor NF-κB activation by LMP1. These HEK293-derived cells express the gene for secreted embryonic alkaline phosphatase (SEAP) under the control of an NF-κB promoter [26]. 80,000 293-SEAP reporter cells, grown in DMEM medium with 10% FBS, were plated in each well of a 96-well plate. Viral stocks of Ad5-LMP1 were serially diluted and added to the reporter cells in triplicate, in the presence or absence of 1ug/ml doxycycline (Alfa Aesar) added at the time of transduction. After 36 h, 10 μl/well of the supernatants was added to the wells of a 96-well assay plate together with 100 μl/well of QUANTI-Blue Alkaline Phosphatase substrate (InvivoGen). The plates were incubated for 20 min at 20°C and OD was read at 650 nm.