FIGURE 6.

Physical interaction of CaMKII with SMRT in intact cells. A, recombinant GST or GST-SMRT was immobilized onto GSH-agarose. HEK293 cells were transfected with 0.5 μg of HA-CaMKII. 48 h after transfection, the cell lysates were subjected to GST pull-down experiments with immobilized GST or GST-SMRT. Proteins bound to GST or GST-Notch1-IC were analyzed via immunoblotting with an anti-HA antibody. 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 MG132 (5 μm) for 6 h. The cell lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody. The immunoprecipitates were then immunoblotted (IB) with an anti-Flag antibody. Cell lysates were also immunoblotted with anti-Flag and anti-HA antibodies. C, HEK293 cells were lysed and subjected to immunoprecipitation with immunoglobulin G (IgG) and anti-SMRT antibodies as indicated. The immunoprecipitates were immunoblotted with an anti-CaMKII antibody. Cell lysates were immunoblotted with anti-SMRT and anti-CaMKII. D, recombinant GST or GST-SMRT deletion proteins (GST-SMRT-SD1, GST-SMRT-SD2, GST-SMRT-RID1, and GST-SMRT-RID2) were immobilized onto GSH-agarose. HEK293 cells were transfected with 0.5 μg of HA-CaMKII. 48 h after transfection, the cell lysates were subjected to GST pull-down experiments with immobilized GST or GST-SMRT. Proteins bound to GST or GST-SMRT were analyzed via immunoblotting with an anti-HA antibody. CBB, Coomassie Brilliant Blue staining; IgG_H, immunoglobulin heavy chain; IB, immunoblot.