Sullivan, C., Postlethwait, J.H., Lage, C.R., Millard, P.J. and Kim, C.H.
Notes: The authors used reporter assays and coimmunoprecipitation experiments to compare the zebrafish (Danio rerio) Toll-IL-1R-containing adaptor molecule 1 (TICAM1) activation of NF-κB and zebrafish type I IFN to mammalian TICAM1 activation. 293H and ZFL (zebrafish liver) cells were cotransfected with 400 ng of TICAM1 construct (mouse, zebrafish or a deletion construct), 400 ng of a reporter construct (e.g., a zebrafish IFN promoter cloned into the pGL3-Basic Vector) and 10 ng of pRL-CMV Vector, an internal control to normalize data. After 24 hours for 293H cells and 48 hours for ZFL cells, the transfected cells were lysed, and luciferase activity was measured using the Dual-Luciferase® Reporter Assay System. For coimmunoprecipitation experiments, 293H cells were cotransfected with a total of 8µg of plasmids (3µg of zebrafish or mouse TICAM1 construct, 3µg of interacting protein construct, 1µg of the pAdVAntage™ Vector, and 1µg of antiapoptotic protein p35 construct). Forty-eight hours posttransfection, the cells were lysed, the protein bound to affinity resins and analyzed by Western blot. (3755)