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Figure 5


Fig. 5. p38 MAPK operates downstream of Dishevelleds. (A) F9 cells expressing Rfz1 were treated with siRNAs designed to suppress the expression of Dvl3 for 48 hours and p38 MAPK activity was assayed after 15 minutes of stimulation with Wnt3a. The extent of suppression of Dvl3 is more than 85%. Upper panel represents mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-ATF2-P (p-ATF2), anti-p38 (p38) and anti-Dvl3 (Dvl3) antibodies. (B) F9 cells stably expressing Rfz1 were transfected with Dvl3-GFP2 for 24 hours and p38 MAPK activation was determined. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-ATF2-P (p-ATF2), anti-p38 (p38) and anti-HA (HA) antibodies. (C) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with 100 nM siRNA specific to mouse Dvl3 for 48 hours and the lysates were assayed for cytosolic β-catenin levels after 4 hours of Wnt3a treatment. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-β-catenin antibody (β-catenin), anti-Dvl3 (Dvl3); immunoblots probed with anti-actin antibody (actin) were used as loading controls. (D) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were transfected with either empty vector (–) or with HA-Dvl3-GFP2. After 4 hours of transfection, the transfection medium was replaced with serum medium (15%) containing either DMSO or SB203580 (6 µM) and cultures grown for an additional 20 hours. After 20 hours, the lysates were collected and luciferase assay was performed. Upper panel represents mean values ± s.e.m. from three independent experiments performed in triplicate; lower panel shows representative blots probed with anti-HA (HA) and anti-actin (actin) antibodies. *P<0.05 and **P<0.01 versus –Wnt3a control; #P<0.05 and ##P<0.01 versus +Wnt3a control.





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