Detection of Thr434 phosphorylation with the help of a phosphorylation site-specific antibody. A, Verification of antibody specificity. – Recombinant SF3b1-NT-His6 (WT) or the T434A point mutant thereof were phosphorylated in vitro by GST-DYRK1A-ΔC or incubated under the same conditions in the absence of GST-DYRK1A-ΔC. The indicated amounts of SF3b1-NT-His6 (500 ng, 100 ng, 20 ng, 4 ng) were separated by SDS-PAGE and subjected to Western blot analysis with antibodies specific for pT434, pThrPro (pTP), and SF3b1. B, Phosphorylation of Thr434 in vivo. – COS-7 cells seeded in 6-well plates were co-transfected with expression plasmids for wild type GFP-SF3b1-NT (WT) or the T434A mutant (0.6 μg/well) and either GFP (Co) or GFP fusion constructs (0.1 μg, 0.2 μg or 0.4 μg/well) of the indicated protein kinases (wild type DYRK1A or the K188R point mutant, DYRK1B, CLK3, HIPK2). The total amount of transfected DNA was kept constant by addition of vector DNA where appropriate. Two days after transfection, total cellular lysates were prepared and subjected to Western blot analysis with antibodies specific for pT434, pThrPro (pTP), SF3b1, or GFP. A shorter exposure of the top panel is shown below because the most intense signals exceeded the linear range of the detection camera. A slowly migrating form of SF3b1-NT is marked by an asterisk (*). C, Nuclei were purified from COS-7 cells transfected with expression plasmids for wild type GFP-DYRK1A (1 μg, 2 μg or 4 μg/6-cm plate) or the point mutant K188R. Nuclear proteins were subjected to Western blot analysis with the indicated antibodies.
de Graaf et al. BMC Biochemistry 2006 7:7 doi:10.1186/1471-2091-7-7