Figure 2

Phosphorylation of SF3b1 by DYRK1A in COS-7 cells. COS-7 cells were transiently transfected with expression plasmids for GFP-SF3b1-NT and the protein kinases DYRK1A or CLK3. Two days after transfection, cells were lysed under denaturing conditions and the recombinant proteins were immunoprecipitated with polyclonal GFP antiserum (A, B, C) or monoclonal SF3b1 antibody (D). Immunoprecipitates were subjected to Western blot analysis with antibodies specific for phosphoThr-Pro (anti pTP), GFP, or SF3b1. A, COS-7 cells were transfected with 1 μg of pEGFP-SF3b1-NT and increasing amounts of pEGFP-DYRK1A (0 μg, 0.2 μg, 1 μg and 2 μg of DNA per 6-cm plate). B, Cells were transfected with pEGFP-SF3b1-NT and either pEGFP-DYRK1A-K188R, pEGFP-DYRK1A (WT), pEGFP (Co), or pEGFP-CLK3. C, Cells were transfected pEGFP-SF3b1-NT and either pEGFP-DYRK1A (DIA) or pEGFP (Co). Cells from one plate were lysed in buffer lacking phosphatase inhibitors, and the lysate was incubated for 1 h at 37°C with 2000 u of calf intestinal phosphatase (CIP) before immunoprecipitation. D, Cells were transfected pEGFP-SF3b1-NT (left lane) or were not transfected. Migration of the immunoprecipitating antibody is indicated (IgG). The asterisk marks a slowly migrating form of SF3b1-NT (see text).