Figure 3

Interrogation of functional roles of Y17 and S407 sites of Dvl3 in assembly of very large Dvl3-based supermolecular complexes in response to stimulation with Wnt3a. Y17D-Dvl3 and S407A-Dvl3 abolished the assembly of Dvl3-based supermolecular complexes in response to Wnt3, whereas S407D-Dvl3 enhances the assembly of Dvl3-based supermolecular complexes. Cells expressing either wild-type Dvl3 or Y17D-Dvl3 or S407A-Dvl3 or S407D-Dvl3 were stimulated with or without Wnt3a for 30 min. Cells were lysed and extracts (20 mg protein) were applied to Sephacryl S-400 gel filtration column (AKTA, GE Health Care). Fractions were analyzed by SDS-PAGE and immunoblotted, subsequently stained with anti-Dvl3 antibody. Dvl3 blots were quantified by the calibrated scanner and results were displayed. The Dvl3-based supermolecular complexes in F9 cells expressing Rfz1 were also displayed as a control. The calculated, relative molecular weight (M _r ) positions from the calibration curve are labeled at the top. The bottom numbers indicate fraction number. Arrow indicates the precise position at which calibration proteins elute from Sephacryl S-400. Results are representative of at least 2 independent experiments.