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Fig. 2. RGS protein overexpression screen. (A) F9 cells transiently transfected with vectors containing Fz1, M50 and the indicated RGS proteins were treated with or without Wnt3a for 8 hours, then collected and subjected to the luciferase assay. The results show that expression of RGS19 alone can significantly attenuate Wnt3a-induced gene transcription by more than 50%. *P<0.05 for the difference between wild-type control cells and those expressing RGS19. Con, control. The data shown are mean values (±s.e.) of at least three separate experiments. (B) Lysates were collected from F9 cells individually transiently transfected with an HA-tagged RGS protein. Each sample was subject to SDS-PAGE on an 11% acrylamide gel, transferred to nitrocellulose and probed with an antibody against HA. (C) Western blot analysis was used to determine the presence of RGS19 at the protein level in F9 cells. Wild-type lysates were separated by centrifugation into crude membrane (Mem) and cytosolic (Cyto) fractions. Each sample was subject to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against RGS19. In agreement with previous reports, one band is seen in the cytosolic fraction and two in the membrane fraction. Each fraction was treated with alkaline phosphatase (AP), then subjected to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against RGS19. P-RGS19, phospho-RGS19.
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