Fig. 6: Wnt responses of cells bearing Axin1 point mutants.
SuperTOP assays and corresponding Western blots of HEK293T lines bearing CRISPR-engineered a DED3 > A, b S4 > A, c RKR > A, d P372A or e RR > D mutations in endogenous Axin1 (in an Axin2 KO background) +/− Wnt3a, as indicated; representative Western blots from the same lysates were probed with antibodies as indicated on the right; below, names of selected mutant lines; open circles, individual values obtained from at least three independent experiments are set relative to the mean value of three samples from Wnt-stimulated parental HEK293T cells (wt, set to 100%); statistical significance was determined by one-way ANOVA with multiple comparisons, comparing each treatment to Wnt-stimulated wt cells (+) following normalization; all p-values are too small to be determined exactly (****, p < 0.0001) except for (a) ***, p = 0.0008 (wt+ vs DED3 c16 + ) or ***, p = 0.0002 (wt+ vs DED3 c32 + ); (e) *, p = 0.0448 (wt+ vs RR > D c18 + ) or ns (not statistically significant), p = 0.6179 (wt+ vs RR > D c18 + ); error bars, ± SEM. f Western blot representative of three independent experiments, showing comparative analysis of selected mutant lines (+/− Wnt3a) shown in (a–e); l.e., longer exposure of the α-Axin1 blot. Note the marked destabilization of DED3 > A and S4 > A observed in each independently isolated mutant line, which may reflect reduced binding of the USP7 de-ubiquitylase to these LIRup mutants (see text).
