Figure 3. APC2 stabilizes Axin complexes and promotes efficient βcat destruction.
(A) Stills, FRAP movie, SW480 cells transfected with GFP-APC2 (shown) and Axin-RFP. Inset = magnified APC2 signal in punctum. (B) APC2 recovers to ∼40% when in Axin puncta. Recovery curve (red); unbleached control (blue). (C) Axin expressed alone plateaus at ∼80%. (D and E) Axin is stabilized when coexpressed with APC2. (F) Total cell βcat fluorescent intensity normalized to untransfected cells (= 100%). APC2 or APC2 + Axin expression lead to stronger βcat reduction than Axin alone. (G–I) Indicated constructs expressed in SW480 cells. Insets = regions boxed. (G) GFP-Axin forms puncta and reduces βcat levels in this hAPC1 mutant cell line. βcat is detectable in puncta (arrows). (H) GFP-APC2 expressed alone is dispersed throughout the cell and βcat levels are low overall and in puncta. (I) Axin-RFP + GFP-APC2 coexpressed. βcat is reduced in APC2:Axin puncta (arrow) relative to puncta with Axin alone (G). (J) GFP-APC2 is recruited into puncta formed by human hAxin1-RFP. (K) Endogenous human hAxin1 co-IPs from SW480 cells with transfected Flag-APC2. Untransfected cells serve as a negative control. (L and M) Phospho-S33/37-βcat levels are more reduced when either APC2 or APC2 + Axin are expressed relative to Axin alone. (L) Immunoblot , transiently transfected SW480 cell extracts, centrifuged at 1000 rpm. (M) Quantification, phospho-S33/37-βcat protein levels from (L) and 2 replicates. Student's t-test.
Figure 3—figure supplement 1. While proteasome inhibition reduces βcat destruction and causes βcat to detectably accumulate in APC2 + Axin puncta, it does not abolish the ability of APC2 to enhance Axin function in this regard.
