FIGURE 5.

CaMKII down-regulates the steady state level of SMRT protein. A, HEK293 cells were transfected with 0.5 μg of Flag-SMRT, 0.5 μg of HA-CaMKII, and 0.5 μg of HA-CaMKII-DN as indicated. 48 h after transfection, the cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. B, HEK293 cells were transfected with 0.5 μg of Flag-SMRT and 0.5 μg of HA-CaMKII as indicated. 42 h after transfection, the cells were treated with various concentrations (0, 1, and 5 μm) of MG132 for 6 h. 48 h after transfection, the cell lysates were immunoblotted with anti-Flag and anti-HA antibodies. C, HEK293 cells were transfected with 0.5 μg of Flag-SMRT, 0.5 μg of HA-CaMKII, and 0.5 μg of HA-CaMKII-DN as indicated. 42 h after transfection, the cells were then treated with 0.1 mm cycloheximide for the indicated times. D, HEK293 cell were treated with the indicated amounts of purified Wnt5a protein for 8 h. The cell lysates were also subjected to immunoblotting analysis with anti-SMRT. Probing with an antibody to β-actin was used as a loading control. E, HEK293 cells were transfected with 0.5 μg of Flag-SMRT, 0.5 μg of HA-ubiquitin, and 0.5 μg of GFP-CaMKII as indicated. 42 h after transfection, HEK293 cells were treated with KN-93 (5 μm) for 6 h. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody and the immunoprecipitates were immunoblotted with anti-HA antibody. The cell lysates were also subjected to immunoblot analysis with anti-HA, anti-Flag, and anti-GFP antibodies, respectively. Probing with an antibody to β-actin was used as a loading control. DN, dominant-negative; IB, immunoblot.