Figure 5

Aldolase activates Wnt signaling by disrupting the Axin-GSK-3β interaction and targeting Axin to the Dvl “puncta”. (A) HEK293T cells were co-transfected with GFP-Axin or an empty vector, and with GFP-ALDOC, GFP-ALDOB or an empty vector. Forty eight hours post transfection, cells were harvested and lysates were co- immunoprecipitated with goat anti-Axin antibody or with control un-immuned serum and separated by SDS-PAGE. Blots were incubated with anti-GFP; anti-GSK-3β or anti-tubulin (B) COS-7 cells were co-transfected with myc-Dvl and FLAG-Axin along with GFP-ALDOB, GFP-ALDOC or an empty vector. Forty eight hours post transfection; cells were fixed, permeabilized and incubated with anti-FLAG and anti-myc antibodies. Cell nuclei were then stained with DAPI, and visualized using a confocal microscopy. SB was used as a positive control for activation of the Wnt pathway (C) Stained cells from B were scored according to Axin distribution in large “puncta” (2 for only large, 1 for large and small, 0 small or diffused distribution) 20-25 cells were scored for each condition.