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Fig. 8. Knockdown of RGS19 protein attenuates Wnt3a-induced Lef-Tcf reporter activation. (A) F9 cells transiently transfected with an expression vector harboring siRNA sequences targeting RGS19 (or a control) and encoding Fz1 and M50 were treated with or without Wnt3a for 8 hours, then collected and subjected to the luciferase assay. The results show that specific knockdown of RGS19 decreases Wnt3a-mediated gene transcription by more than 50%. *P<0.05 for the difference between cells transfected with the control siRNA vector and those transfected with the siRNA vector targeting the expression of RGS19. (B) Western blot analysis was used to measure the effect of siRNA treatment on RGS19 protein levels. Lysates were collected from cells transfected with each siRNA vector, then subjected to SDS-PAGE on a 12.5% acrylamide gel, transferred to nitrocellulose and probed with an antibody against RGS19. The siRNA targeting RGS19 was able to lower RGS19 protein expression to less than 10% that of control (Con) cells. 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 an expression vector harboring siRNA sequences targeting the expression of RGS19 (or a control) were treated with Wnt3a for the indicated lengths of time. Lysates were collected and treated with Con A to separate cytosolic from membrane-associated 
-catenin. Cleared samples were subjected to SDS-PAGE on an 11% acrylamide gel, transferred to nitrocellulose and probed with an antibody against -catenin. The increase in cytosolic -catenin that is seen in response to Wnt3a is attenuated by knockdown of RGS19. (D) F9 cells transiently transfected with vectors encoding Fz1, M50, the indicated amounts of RGS19 and an expression vector harboring siRNA sequences targeting the expression of RGS19 were treated with Wnt3a for 8 hours, then collected and subjected to the luciferase assay. The data shown are mean values (±s.e.) of at least three separate experiments. *P<0.05 for the difference between cells transfected with the control siRNA vector and those transfected with the siRNA vector targeting expression of RGS19 alone or in combination with 0.5 µg or 1 µg of RGS19.
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