Fig. 1.

DHX32 interacts with β-catenin and protects β-catenin from degradation. (a) Flag-tagged DHX32 transiently expressed in SW480 cells was purified and applied to SDS-PAGE. The proteins were visualized by silver staining, and indicated spots were analyzed by mass spectrometry. (b) Interaction of endogenous DHX32 and β-catenin proteins. Cell lysates from SW480 cells were subjected to IP with antibody against DHX32 followed by immunoblotting (IB) to detect endogenous β-catenin. (c) In vitro interaction between DHX32 and β-catenin. Bacterial-expressed and purified His-tagged DHX32 and GST-tagged β-catenin were subjected to GST pull-down assays as indicated. DHX32 proteins were detected with anti-DHX32 antibody. GST and GST/β-catenin were determined by Ponceau S staining. (d) Endogenous β-catenin protein levels in SW480 cells with DHX32 depletion or overexpression were detected by immunoblotting. (e) DHX32 decreases ubiquitination of β-catenin. After overnight treatment of MG-132 (10 μM), SW480 stable cells were subjected to anti-β-catenin IP. The ubiquitin-conjugated β-catenin ((Ub)_n-β-catenin) was detected with anti-Ub antibody. (f) DHX32 regulates turnover rates of β-catenin. The levels of β-catenin at different time points after cycloheximide (CHX) treatment with SW480 stable cells were determined by immunoblotting the total cell lysates and quantification using Image Lab software (Bio-Rad) with β-Actin as a loading control. Results plotted are the amounts of β-catenin at each time point relative to the level at time 0. (g) DHX32 colocalizes with β-catenin proteins at nucleus. Localization of DHX32 (red) and β-catenin (green) in SW480 cells with DHX32 overexpression or depletion were detected by immunofluorescence. The nuclei were stained with DAPI (blue).