Fig. 6. p38 suppresses GSK3β activity. (A) Confluent F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with either vehicle (DMSO) or SB203580 (6 µM) for 1 hour prior to the addition of Wnt3a for indicated periods of time. After stimulation, the lysates were collected and subjected to immunoblot analysis with anti-GSK3β Ser9-P antibody. Upper panel represents mean values ± s.e.m. from three independent experiments and the lower panel represents representative blots probed with either anti-GSK3β Ser9-P (p-GSK3β Ser 9) or with anti-GSK3β (GSK3β) antibodies. (B) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were either transfected with empty vector (–) or with Flag-tagged dominant-negative (DN) mutant of p38 MAPK [p38 (AGF)] for 24 hours. After Wnt3a stimulation for 1 hour, the lysates were collected and subjected to immunoblot analysis with anti-GSK3β Ser9-P antibody as described. Upper panel represents mean values ± s.e.m. from three independent experiments and the lower panel represents representative blots probed with either anti-GSK3β Ser9-P (p-GSK3β Ser 9), anti-p38 (p38) or with anti-GSK3β (GSK3β) antibodies. (C) Confluent F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with either vehicle (DMSO) or SB203580 (6 µM) for 1 hour prior to the addition of Wnt3a for 10 minutes. GSK3β was immunoprecipitated from whole cell lysates and its activity was measured by an in vitro kinase assay. The data represent mean values ± s.e.m. from two independent experiments that are highly reproducible. ^*P<0.05 and ^**P<0.01 versus –Wnt3a control; ^#P<0.05 and ^##P<0.01 versus +Wnt3a control.