Figure 2. Dvl2-Grb2 interaction involves the Dvl2 proline-rich region (PRR)-2.

(A) Site-directed mutagenesis was used to construct ΔPRRs from wild type (WT) Dvl2-FLAG, deleting the indicated sequences. (B) Mutant forms of Dvl2 are expressed equivalently to WT. Whole cell lysates were harvested from transfected HEK293s and western blotted using an anti-FLAG antibody. Blots were stripped and re-probed using or anti-tubulin antibodies as a loading control. (C) Loss of PRR1 or PRR2 reduces Dvl2-Grb2 interaction. FLAG-tagged WT or Dvl2 PRR mutants were co-transfected along with HA-Grb2 and immunoprecipitation was performed using anti-FLAG antibody as described in Fig. 1. Strips were cut from the same blot and were probed with anti-FLAG or anti-HA antibodies. (D) Synergy between Dvl2 and Grb2 depends on PRR2 but not PRR1. HEK293 cells were transfected with plasmids encoding WT or mutant Dvl2 along with Grb2 or GFP and sTF. Cells were harvested for luciferase activity at 24 hours. * – p<0.01 compared to WT Dvl2. All luciferase data are normalized to total protein content. Mean and standard error of the mean are shown. Significance assessed by Student's t-test.