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Fig. 3. RGS19 overexpression attenuates Wnt3a-induced Lef-Tcf reporter activation. (A) F9 cells transiently transfected with vectors containing Fz1, M50 and RGS19 were treated with Wnt3a for the indicated lengths of time, then collected and subjected to luciferase assay. Overexpression of RGS19 (+ RGS19) blocked Wnt3a-induced reporter activation in a time-dependent manner. *P<0.05 for the difference between wild-type, Wnt3a-treated cells and those expressing RGS19 and Wnt3a-treated for the indicated time points. RLU, relative luciferase units. (B) F9 cells transiently transfected with vectors containing Fz1, M50 and the indicated amounts of RGS19 were treated with or without Wnt3a for 8 hours, then collected and subjected to the luciferase assay. The results show that increasing levels of RGS19 can block the Wnt3a-induced reporter activation. *P<0.05 for the difference between wild-type, Wnt3a-treated cells and those expressing RGS19 and Wnt3a-treated for the indicated time points. **P<0.05 for the difference between wild-type, untreated cells and those expressing RGS19 for the individual time points. Lower panel: immunoblotting (IB) western blot analysis was used to measure the expression of transiently transfected HA-tagged RGS19. Lysates were collected from cells transfected with the indicated amounts of HA-RGS19, then subjected to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against HA. The expression of actin was used as a loading control. The results displayed are data from single experiments, representative of more than three independent tests. (C) F9 cells transiently transfected with vectors containing Fz1, M50 and the indicated amounts of RGS17 or RGS19 were treated with or without Wnt3a for 8 hours, then collected and subjected to luciferase assay. RGS19 suppressed Wnt3a signaling in a dose-dependent manner, whereas overexpression of RGS17 had little effect. Results are displayed as the difference in reporter activation upon Wnt3a stimulation (means ± s.e.) as compared with untransfected control and are representative of three independent experiments. *P<0.05 for the difference between wild-type cells and those expressing RGS19 at each concentration. (D) IB western blot analysis was used to measure the expression of transiently transfected HA-tagged RGS19 and RGS17. Lysates were collected from cells transfected with the indicated amounts of HA-RGS19 and HA-RGS17, then subjected to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against HA.
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