Figure 5. Dvl-based complexes are heterogeneous with respect to Dvl isoforms.

A, Dvl isoform-specific antibodies were tested for cross reactivity by immunoblotting of whole-cell lysates obtained from cell “over”expressing a single Dvl isoform. Immunoblots of cell lines that were “over”expressing a single Dvl isoform then were stained with individual antibodies against each of the Dvl isoforms. B, immune precipitation-based “pull-downs” from whole-cell lysates of F9 cells were performed using antibodies that recognize only Dvl2. The Dvl-based complexes isolated in the pull-downs were subjected to SDS-PAGE and the resolved proteins transferred to nitrocellulose blots. The blots were stained with antibodies specific for each Dvl isoform, seeking to detect Dvl1, DVl2, and Dvl3 that may be present in the complex. C, immune precipitation-based “pull-downs” from whole-cell lysates of HEK293 cells were performed using antibodies that recognize only either Dvl2 or Dvl3. Whole-cell lysates (“total”), pull-down complexes (“IP”) as well as supernatant fraction following the immune precipitation reaction (“post-IP”) were subjected to SDS-PAGE and the resolved proteins transferred to nitrocellulose blots. The blots were stained for each Dvl isoform. D, HEK293 cells expressing Fz1 were treated without and with purified Wnt3a for up to 60 min and the composition of Dvl2-based as well as Dvl3-based complexes analyzed for the presence of all three isoforms in the complex performed by immunoblotting. Immune precipitation-based “pull-downs” were performed using antibodies that recognize either Dvl2 or Dvl3. Whole-cell lysates (total), pull-down complexes (IP) as well as supernatant from post-immunoprecipitated fraction (post-IP) were subjected to SDS-PAGE and the resolved proteins transferred to nitrocellulose blots and stained for each Dvl isoform. The data presented in these panels are blots representative of three or more independent analyses for each protocol. The data from each of the experiments are in agreement.