Figure 1.

(a) GBP blocks GSK-3 binding to Axin in vivo. Embryos were injected at the two to eight-cell stage in the animal pole with 50 pg Xgsk-3-FLAG and 1 ng Axin-myc along with increasing doses of GBP-myc, as indicated. After 4–5 h, proteins were isolated by immunoprecipitation with anti-FLAG antibody and detected by Western blotting (left panel). An aliquot of each sample taken before immunoprecipitation is shown in the right panel (Total Lysates); the level of Xgsk-3 produced is not detectable in these samples (∼1 embryo). A portion of the total lysate shown in lane 6 was precipitated in the absence of anti-FLAG antibody as a negative control (lane 7). Lane numbers in the right panel refer to the same injections as shown above the corresponding lane numbers in the left panel. (b) Prevention of GSK-3–Axin binding by GBP is dependent on GBP/GSK-3 binding. Embryos were injected with Axin-myc and Xgsk-3-FLAG as above along with 4 ng HA-tagged wild-type (GBPwt) or the HA-tagged point mutant that does not bind GSK-3 (GBPmut). Immunoprecipitations were performed as above. No GBP is present in the anti-FLAG immunoprecipitates in lane 4 since this is the GSK-3 binding mutant of GBP. Lane numbers in the right panel refer to the same injections as shown above the corresponding lane numbers in the left panel.