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Fig. 6. Expression of dominant-negative (DN) versions of either MEKK 1 or MEKK 4 as well as use of siRNAs specifically targeted to MEKK 1/MEKK 4 blocks the activation of JNK in response to WNT3a stimulation. (A,B) F9 cells expressing rFZ1 were transfected (1 µg/well in a 12-well plate) with an expression vector harboring either DN-MEKK 1 or DN-MEKK 4 (A) or treated with siRNAs specific to either MEKK 1 or MEKK 4 (B) for 24 hours, and JNK activity was determined. (C) Confluent F9 cells expressing rFZ1 were treated with or without inhibitors of JNK (SP600125, 0.4 µM) or p38 (SB203580, 6 µM) for 1 hour before stimulation with WNT3a, followed by determination of JNK activity. (D) F9 cells stably expressing rFZ1 were transfected with DVL3-GFP2 either alone or together with DN-MEKK 1 for 24 hours, followed by determination of JNK activity. (E) F9 cells stably expressing rFZ1 were transfected with constitutively active (CA)-Cdc42 either alone or together with DN-MEKK 1 for 24 hours, followed by determination of JNK activity. The upper panel displays mean values±s.e.m. obtained from three independent experiments; the lower panel displays the corresponding representative blots. The DVL3–DN-MEKK 1 and CA-Cdc42–DN-MEKK 1 epistasis experiments are representative of two independent experiments whose results were in strong agreement. *P<0.05; **P<0.01; versus –WNT3a control; #, P<0.05; ##, P<0.01 versus +WNT3a control.
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