Figure 8. The 20R2 of APC is required to inhibit β-catenin transcriptional activity (A) and to down-regulate β-catenin (B) but was not necessary to recruit Axin in the presence of SAMP repeats (C).

A, SW480 cells were transiently transfected with control vector (flag) or the indicated APC constructs, fixed on day 3 and stained with an anti-β-catenin antibody. Bar, 10 μM. C, DLD1 cells were transiently transfected on day 1 with the indicated APC constructs or N-terminal flag-tagged Axin, either individually or in combination. The cells were fixed on day 3 and were stained with an anti-flag antibody. Bar, 10 μM. B, SW480 cells were transiently transfected on day 1 with reporter plasmids and 100 ng of empty vector (flag) or the indicated APC constructs. yAPC-2μ contains the mutations illustrated in figure 4A. TOP/FOP reporter assays were performed on day 3 to measure β-catenin transcriptional activity. The data are presented as the mean ± standard deviation of three independent values from a representative experiment. * p<0.0001 compared with yAPC, Student's t-test. In a parallel experiment, cells were transiently transfected with 1 μg of the indicated plasmids on day 1. Cell extracts were prepared on day 3 and were subjected to western blotting using the indicated antibodies. The molecular weights are presented in kDa. C, DLD1 cells were transiently transfected on day 1 with the indicated APC constructs or N-terminal flag-tagged Axin, either individually or in combination. The cells were fixed on day 3 and were stained with an anti-flag antibody. Where applicable, the percentages indicate the proportion of different localisation patterns in the transfected cells. The imaging parameters were identical for each type of tag. Bar, 10 μM.