Fig. 4

The two isoforms of EDARADD exhibit different activities and stabilities in cellulo. a Constructs. HA constructs: HA tag was N-terminally fused to EDARADD A or B protein. Myc constructs: Myc tag was C-terminally fused to EDARADD A or B protein. These constructs were generated using recombination so that 1A and 1B constructs only differ for A and B peptide coding region. NF-κB reporter gene: NF-κB are located upstream a minimal promoter and a luciferase reporter gene. b EDARADD A and B activate the NF-κB pathway in a dose-dependent manner. Increasing amounts of constructs encoding A or B isoforms were transfected in HEK cells with NF-κB reporter plasmid that harbors NF-κB response elements upstream luciferase reporter gene. The luciferase activity was normalized against β-gal and the empty vector. The luciferase activity was assayed 24 hours after transfection. In yellow: isoform A, in brown: isoform B. c A isoform is twice more active than B isoform when luciferase activity is corrected for Edaradd quantity. HEK cells were transfected with constructs encoding EDARADD A or B C-terminally tagged with myc (EDD-myc A or B) and the luciferase reporter gene as in A. 24 h after transfection, three wells were pooled in order to have enough material for Western Blotting and three others were used for luciferase assays. Proteins were detected by Western Blot with an anti-Myc antibody. Histone H3 was used as a control. Luciferase activity corrected for edaradd quantity is presented on the right. d EDARADD A and B reach different steady state levels. HEK cells were transfected with constructs encoding EDARADD A or B N-terminally tagged with HA (HA-EDD A or B) or C-terminally tagged with myc (EDD-myc A or B). Proteins were detected by Western Blot 12 h, 24 h, 36 h and 48 h after transfection. Histone H3 was used as a control. e EDARADD A but not B is destabilized by NF-κB activators. Western Blot for Myc C-terminally-tagged A and B isoforms following co-transfection of NF-κB activators such as EDAR or TRAF6, at 24 or 48 h. Histone H3 is used as a control