Downregulation of endogenous DYRK1A reduces phosphorylation of Thr434 in SF3b1. A, Test of shRNA vectors for DYRK1A knockdown. – HEK293T cells seeded in 6-well plates were co-transfected with expression plasmids for GFP-DYRK1A (0.2 μg/well) and either empty vector (Ctrl) or plasmids expressing to different small hairpin RNAs directed against DYRK1A (673 or 2060; 0.8 μg DNA/well). Two days after transfection, nuclear extracts were prepared and subjected to Western blot analysis with a DYRK1A-specific antibody. B, Effect of DYRK1A knockdown on Thr434 phosphorylation. – HEK293T cells or HepG2 cells were co-transfected with the expression plasmid for GFP-SF3b1-NT (0.5 μg/well) and the pSUPER constructs (0.8 μg/well). Total cellular lysates were subjected to Western blot analysis with antibodies specific for pThr434 or GFP. The asterisks mark unspecific bands (*).