Effect of RasGrf1 on mPttg1 promoter activity. Luciferase reporter assays carried out in insulinoma BTC3 cells cotransfected with plasmid pGL3 (containing a 2,3 Kb promoter region of mPttg1) plus one additional construct, corresponding either to empty plasmid vector pBKCMV (black boxes) or to construct pBKCMV-RasGrf1, expressing a full length RasGrf1 cDNA clone (white boxes). (A, B) Assays were performed 48 hours after cotransfection and included treatment for 7 hours with the indicated agonists (panel A) or inhibitors (panel B) of signaling pathways, under conditions detailed in Methods. (C) Western immunoblot showing level of overexpression of the RasGrf1 full length protein in BT3 cells after 48 h transfection with pBKCMV-RasGrf1. Error bars indicate coefficient of variation. * p < 0.05; ** p < 0.01; n = 3 for Vector and RasGrf1 samples. Dotted lines: comparison between RasGrf1 overexpression and transfection with the control vector. Solid black lines: comparison between treated and non treated cells.