Fig. 5. A20 inhibits LPA-mediated NF-B activation. (A) MEFs were infected in 100 mm Petri dishes with an empty retroviral construct or construct expressing hemagglutinin (HA)-flagged forms of A20wt and A20mut and subsequently transfected with a luciferase reporter plasmid for NF-B. Infected cells were left untreated or stimulated with LPA (20 µM) for 8 hours and luciferase activity was measured. The data shown represent the relative luciferase activity and are representative of four independent experiments performed in triplicate. (B) MEFs were infected as in panel A and left untreated or treated with LPA for 60 minutes. Lysates were then prepared and immunoprecipitated (IP) with antibody against IKK/NEMO. Immunocomplexes were separated by SDS-PAGE, transferred onto membranes and subsequently probed with antibodies (WB) against BCL10 and IKK/NEMO. A20 expression was monitored by immunoblotting with antibody against HA.