G_o mediates WNT-JNK signaling through Dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells

JCS021964 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Effect of WNT5a on activation of Jun NH2-terminal kinase in F9 cells. F9 cells stably expressing rat Frizzled-1 (rFZ1) were treated with purified WNT3a (20 ng/ml) or WNT5a (25 ng/ml, R&D Systems, Minneapolis, MN) for 15 minutes, and JNK activation was measured by immunoblotting total lysates with phospho-specific antibodies against Jun. The data represent two independent experiments performed in duplicate, with the results being in strong agreement with one another.

• Supplemental Figure S2 -

  Fig. S2. Activation of both WNT3a-JNK and WNT3a-canonical-pathways in wild-type HEK 293 cells. (A) Confluent HEK 293 cells were treated with purified WNT3a (20 ng/ml) for 15 minutes, and JNK activation was measured by immunoblotting (IB) total lysates with phospho-specific antibodies against Jun. (B) HEK 293 cells were transfected with the pTOPFLASH luciferase reporter gene in 12-well plates for 24 hours, followed by serum starvation overnight. The cells then were treated with purified WNT3a (20 ng/ml) for 7 hours and then lysed. 20 µl of the lysate was added to 100 ml of luciferase assay buffer (20 mM Tricine, pH 7.8, 1.07 mM MgCO₃, 4 mM MgSO₄, 0.1 mM EDTA, 0.27 mM coenzyme A, 0.67 mM luciferin, 33.3 mM DTT and 0.6 mM ATP), and the luciferase activity was measured by use of a luminometer (Berthold Lumat LB 9507). (C) Total RNA from HEK 293 cells was isolated and RT-PCR was performed using human Frizzled-specific primers. The data are from two independent experiments that were in strong agreement. Marker (Mar): 1 Kb plus DNA ladder, (−) no RT control.

• Supplemental Figure S3 -

  Fig. S3. Effect of overexpression of DVL proteins on activation of N-terminal kinase (JNK) in F9 cells. F9 cells expressing rFZ1 were transfected with an expression vector harboring HA and GFP2-tagged versions of DVL1, DVL2 and DVL3 (0.5 µg/well in a 12-well plate) for 24 hours. The JNK activity was then determined, as described in Materials and Methods. The expression of the exogenous DVL proteins is made visible by probing with antibodies against HA. The upper panel displays mean values±s.e.m. obtained from three independent experiments; the lower panel displays the corresponding representative blots.*P<0.05; **P<0.01 versus the no-plasmid control.

• Supplemental Figure S4 -

  Fig. S4. Effect of overexpression of constitutively active mutants of low-molecular-mass GTPases on activation of Jun N-terminal kinase (JNK) in F9 cells. F9 cells expressing rFZ1 were transfected (1 µg/well in a 12-well plate) with constitutively active forms of RhoA, Rac1 or Cdc42 for 24 hours. The JNK activity then was determined, as described in Materials and Methods. The upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays the corresponding representative blots. *P<0.05; **P<0.01 versus the no-plasmid control.