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Figure 2


Fig. 2. Suppression of G{alpha}o, but not G{alpha}q or G{alpha}11, abolishes, whereas expression of constitutively active G{alpha}o mimics, the activation of JNK by WNT3a. (A) Confluent F9 cells expressing rFZ1 were treated with pertussis toxin (50 ng/ml) for 1 hour followed by WNT3a treatment for 15 minutes. Cell lysates were collected and JNK activity was assayed by probing Jun phosphorylation. (B-D) F9 cells expressing rFZ1 were treated with siRNAs specific for G{alpha}o (B), G{alpha}q (C) or G{alpha}11 (D) for 72 hours before treatment with WNT3a for 15 minutes. JNK activity was then determined by probing Jun phosphorylation. (E) F9 cells expressing rFZ1 were transfected with an expression vector harboring the Q205L G{alpha}o mutant (0.25 µg/well in a 12-well plate). 48 hours after transfection, cells were treated with WNT3a for 15 minutes, cell lysates collected and probed with phospho-Jun-specific antibodies. The upper panel displays mean values±s.e.m. obtained from three independent experiments; the lower panel displays corresponding representative blots. *P<0.05; **P<0.01 versus the –WNT3a control; #, P<0.05; ##, P<0.01 versus the +WNT3a control. The extent of knockdown of the expression of the G-protein alpha-subunit routinely was 70% or more.





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