Figure 2

Interrogation of the functional status of Y17 and S407 sites of Dvl3 in Lef/Tcf-sensitive transcriptional activation in response to stimulation with Wnt3a. Panel A, F9 cells co-expressed with Rfz1, Super8xTOPFlash (M50) and either wild-type Dvl3 or one of following Dvl3 mutants (Y17D-Dvl3, S407A-Dvl3 and S407D-Dvl3) were stimulated with or without Wnt3a for 7 hr. Cell lysates were assayed for Lef/Tcf-sensitive transcription. Cell lysates were immunoblotted and subsequently stained with anti-GFP (Dvl3 expression) and anti-GAPDH (control) antibody. Results are displayed for transcriptional activity relative to unstimulated cells (set to “1”). Statistical significance is indicated (*, p < 0.005, versus Dvl3 expressed cells). Panel B, F9 cells stably expressing Rfz1 and M50 were treated with siRNA targeting Dvl3 one day before transfection of the cells with either wild-type Dvl3 or Y17D-Dvl3 or S407A-Dvl3 or S407D-Dvl3. On the following day, cells were stimulated with Wnt3a for 7 hr. Cell lysates were assayed for Lef/Tcf-sensitive transcription. Cell lysates were immunoblotted with either anti-GFP or anti-GAPDH antibody (control). Results are displayed for transcriptional activity relative to unstimulated cells (set to “1”). Statistical significance is indicated (*, p < 0.05, versus Dvl3 expressed cells).