Figure 2
Thr286 phosphorylation mediates the effect of TSA on GFP-cyclin D1 cytoplasmic accumulation A, Thr-286 but not Thr-288 is required for TSA induced cyclin D1 nuclear exclusion. MCF-7 cells were transfected with vectors encoding GFP-Cyclin D1WT, GFP-Cyclin D1T286A, GFP-Cyclin D1T288A or GFP-Cyclin D1T286,-288A for 24 h and treated as for Figure 1. GFP-Cyclin D1 localization was scored by quantitative fluorescence microscopy counting >200 cells in three separate experiments. B, Effect of MG132 on wild type and mutant GFP-Cyclin D1 protein levels. Cells were transfected as in A and then treated with MG132 (50 μM) for 6 h. Cell lysates were separated by 4–20 % SDS-PAGE and immunoblot analysis was done using antibodies against GFP and actin.