Fig. 5. Prolines within the TRAF6 MATH domain are required for morphological changes and activation of both PI 3-kinase and NF-B. (A) HEK293 cells were co-transfected with a PH-Akt-GFP expression vector reporter and various TRAF6 constructs (as described in Fig. 4A) for 24 hours. (B) An NF-B-dependent luciferase reporter containing four tandem B-binding sites adjacent to a minimal fos promoter was assayed 24 hours following cell transfection with either 150 ng of various TRAF6 expression vectors or control vector into HEK293 cells as described in the Materials and Methods. The PI 3-kinase inhibitor LY294002 (Ly) was added at a final concentration of 25 µM into cells after 2 hours of transfection and maintained until analyzed for luciferase activity. Numbers in parentheses indicate fold activity over control vector.