FIG. 3.

Competition of Idax and Axin for their binding to Dvl. (A) Requirement of the DIX and PDZ domains of Dvl for its binding to Axin. After GST–Dvl-1-(1-250) (lanes 1 and 4), GST–Dvl-1-(1-378) (lanes 2 and 5), or GST–Dvl-1-PDZ (lanes 3 and 6) (0.5 μM) was incubated with MBP-rAxin (lanes 1 to 3) or MBP (lanes 4 to 6) (0.5 μM), the GST fusion proteins were precipitated by centrifugation and the precipitates were probed with the anti-MBP antibody. MBP-rAxin (lane 7) and MBP (lane 8) (0.5 μg) were stained with Coomassie brilliant blue. (B) Competition of Idax and Axin for their binding to Dvl in vitro. MBP-rAxin (1 μM) was incubated with GST–Dvl-1-(1-378) (5 pmol) immobilized on glutathione Sepharose 4B in the presence of the indicated concentrations of MBP-Idax (lanes 1 to 5) or MBP (lane 6). GST–Dvl-1-(1-378) was precipitated by centrifugation, and the precipitates were probed with the anti-MBP antibody (upper panel). The amounts of GST–Dvl-1-(1-378) precipitated in each binding assay are shown (middle panel). In reciprocal experiments, MBP-Idax (2 μM) was incubated with GST–Dvl-1-(1-378) (5 pmol) immobilized on glutathione Sepharose 4B in the presence of the indicated concentrations of MBP-rAxin (lanes 7 to 11) or MBP (lane 12). The results shown in the upper and middle panels are representative of three independent experiments. The amounts of MBP-rAxin and MBP-Idax were analyzed using the NIH Image system and expressed as arbitrary units. The results shown are the mean ± the standard error of three independent experiments (lower panel). (C) Inhibition of the binding of Idax to Dvl by Axin in intact cells. The lysates (20 μg of protein) of L-Idax cells transfected with empty vector (lane 1), pEGFPC1-Dvl-1 (lane 2), or pEGFPC1-Dvl-1 and pBJ-Myc/rAxin (lane 3) were probed with the anti-Myc, anti-GFP, and anti-HA antibodies. The same lysates (150 μg of protein) were immunoprecipitated with the anti-GFP antibody, and the immunoprecipitates were probed with the anti-Myc, anti-GFP, and anti-HA antibodies (lanes 4 to 6). IP, immunoprecipitation; Ab, antibody. The results shown are representative of three independent experiments.