FIG. 4.

The downregulation of β-catenin by K5 is proteasome dependent and does not induce Wnt targets. (A) β-catenin is absent upon transduction with AdK5 but not AdK3. E-DMVECs were transduced for 24 h with a high MOI (250) of AdK5 (with AdTet) or AdK3 (with AdTet). K3 and K5 were visualized with anti-FLAG, and β-catenin was also visualized. (B) Reduction of steady-state levels of α-, β-, and γ-catenins in the presence of K5. E-DMVECs were transduced as described in the legend to panel A and processed for intracellular flow cytometry using the indicated antibodies. Dashed line, background staining with secondary antibody only; gray shading, E-DMVECs transduced with AdTet; solid line, E-DMVECs transduced with AdK5 or AdK3. (C) Increase of β-catenin steady-state levels in the presence of proteasomal inhibitors. E-DMVECs were transduced with AdTet (gray shading) or AdK5 (solid line), treated with ConA (50 nM) or MG132 (50 μM) for 6 h, and subjected to intracellular staining for β-catenin. (D) Immunoblot of the indicated proteins upon the transduction of E-DMVECs with the AdTet control or AdK5 (with AdTet). The bottom two rows are from a different experiment. (E) Costaining of K5 and β-catenin in KSHV-infected E-DMVECs. (Upper panels) Latently infected E-DMVECs were stained for spontaneous K5 expression using K5-specific monoclonal antibody 12G6 (unpublished data), together with anti-β-catenin. (Lower panels) Costaining for LANA-1 and β-catenin.