Figure 3.

SCF^FBXO16‐mediated proteasomal degradation of β‐catenin is independent of Wnt/EGF signaling. (A) Whole‐cell protein extracts of MDA‐MB‐231 cells expressing either vector control or DDK‐FBXO16 or DDK‐ΔF‐FBXO16 for 48 h were immunoblotted for the indicated proteins (n = 3). (B) MDA‐MB‐231 cells were coexpressing GFP‐β‐catenin either with DDK‐FBXO16 or DDK‐ΔF‐FBXO16 for 36 h. Transfected cells were then grown in the presence of 5 μm MG132 for 6 h. Whole‐cell lysates were immunoprecipitated with anti‐DDK antibody. Immunoprecipitates and input protein extracts were resolved in SDS‐PAGE and immunoblotted for the indicated proteins (n = 3). (C) MDA‐MB‐231 cells were transfected with the indicated plasmids, including TOP/FOP and pRL‐TK. Transfection efficiency was normalized using the pRL‐TK reporter. Luciferase activity was measured at 48 h posttransfection. After 36 h of transfection, cells were treated with Wnt3a for 12 h. Luciferase activity is shown as a ratio of TOP and FOP. ‘ns’ means statistically not significant (n = 3). (D) Nuclear extracts of MDA‐MB‐231 cells expressing either vector control or DDK‐FBXO16 for 48 h were immunoblotted for the indicated proteins. At 36 h posttransfection, cells were treated with Wnt3a for 12 h (n = 4). (E) Whole‐cell lysates of MDA‐MB‐231 cells expressing either vector control or DDK‐FBXO16 in the absence and presence of EGF for the indicated time periods were immunoblotted for the indicated proteins. (F) MDA‐MB‐231 cells were transfected with the indicated plasmids, including TOP/FOP and pRL‐TK. At 36 h posttransfection, cells were treated with EGF for 12 h, (n = 3). Luciferase activity was measured at 48 h posttransfection, and transfection efficiency was normalized using the pRL‐TK reporter. Luciferase activity is shown as a ratio of TOP and FOP (n = 3).