Figure 7

Expression of Axin M3 mutant precludes assembly of very large Dvl3-based supermolecular complexes in response to Wnt3a. Control cells, Wnt3a stimulates formation of very large M _r -complexes. SEC chromatogram of Dvl3-based complexes prepared from untreated cells. Axin-deficient cells, knockdown of Axin attenuates assembly of Dvl3-based supermolecular complexes in response to Wnt3a. F9 cells were transfected with siRNA targeting Axin one day before transfection with Rfz1. Twenty four hr later, cells were either unstimulated or stimulated with Wnt3a for 30 min. Axin expression, expression of wild-type Axin alone stimulates assembly of Dvl3-based supermolecular complexes. F9 cells were co-transfected with Rfz1 and expression vectors harboring human wild-type Axin. Two days post transfection, F9 cells were treated either without or with Wnt3a for 30 min. Polymerization-defective Axin expression, expression of M3 Axin mutant blocks assembly of very large Dvl3-based supermolecular complexes in response to Wnt3a. F9 cells were co-transfected with Rfz1 and expression vectors harboring human M3 Axin mutant. Two days post transfection, F9 cells were treated either without or with Wnt3a for 30 min. Rescue of Axin-depletion by expression of wild-type Axin, expression of wild-type Axin rescues the inability of Axin-deficient cells to assemble Dvl3-based supermolecular complexes in response to Wnt3a. Cells were treated with siRNA targeting Axin one day before subsequent co-transfection with Rfz1 and human wild-type Axin for an additional day. Twenty four hr after this final transfection, cells were either unstimulated or stimulated with Wnt3a for 30 min. Axin rescue by polymerization-defective Axin, expression of M3 Axin fails to rescue the inability of Axin-deficient cells to assembly of Dvl3-based supermolecular complexes in response to Wnt3a. Cells were treated with siRNA targeting Axin one day before subsequent co-transfection with Rfz1 and M3 Axin mutant for an additional day. Twenty four hr after this final transfection, cells were either unstimulated or stimulated with Wnt3a for 30 min. For all experiments, cell lysates were subjected to Sephacryl S-400 gel filtration column chromatography (SEC) and resolved fractions were analyzed by SDS-PAGE, immunoblotting and eventual staining with anti-Dvl3 antibody. Dvl3 blots were subjected to quantification by calibrated scanning and the results displayed are representative of at least 2 independent experiments, which yielded quantitatively similar results. The calculated, relative molecular weight (M _r ) positions from the calibration curve are labeled at the top of each SEC chromatogram. The bottom labels indicate fraction number. Arrows indicate the precise position at which calibration protein elute from Sephacryl S-400.