Fig. 2.

DYRK2 phosphorylates Notch1-IC. a HEK-293T cells were transfected to express Myc-Notch1-IC and increasing amounts of Flag-DYRK2 wild type (WT) or kinase mutant (KM). Cell lysates were analysed by immunoblotting with the indicated antibodies. We show a representative blot of four independent experiments. b HEK-293T cells were transfected to express DYRK2 WT or KM. Twenty-four hours post-transfection, cells were lysed and protein expression was analysed by immunoblot with the indicated antibodies. We show a representative blot of three independent experiments. c HEK-239T cells were co-transfected with the indicated plasmids and then treated or not for 12 h with the proteasome inhibitor MG-132 (10 μM). Cell lysates were analysed by immunoblotting with anti-Myc and Flag antibodies. We show a representative blot of three independent experiments. d HEK-293T cells were transfected with the indicated plasmids and treated with MG-132 for 12 h and were lysed in phosphatase inhibitor-free buffer in the absence or presence of λ-phosphatase. Electrophoretic mobility was determined by immunoblotting. We show a representative blot of four independent experiments. e Immunoprecipitated Notch1-IC endogenous protein from HEK-293T cells was incubated with DYRK2 recombinant protein in the presence or absence of ATP (0.1 μM). Electrophoretic mobility was determined by immunoblotting with the indicated antibodies. We show a representative blot of four independent experiments. f HEK-239T cells were co-transfected with the indicated plasmids and after 36 h treated with MG-132 in the presence or not of harmine for 12 h before lysis. Protein expression was analysed by immunoblot with the indicated antibodies. We show a representative blot of three independent experiments. g Cells were transfected to express HA-Notch1-IC WT or HA-Notch1-IC T2512A (threonine 2512 mutated to alanine) in the presence or not of Flag-DYRK2 WT. Cells were further cultivated and lysed and protein expression was analysed by immunoblot with the indicated antibodies. We show a representative blot of three independent experiments. h HEK-293T cells were transfected to express Myc-Notch1-IC WT or Myc-Notch1-IC T2512A in the presence or not of Flag-DYRK2-WT or KM and, after 36 h, treated with MG-132 for 8 h and lysed. A fraction was subjected to immunoprecipitation (IP) using anti-Myc antibody. After elution phosphorylation was revealed with an anti-phospho-serine/threonine antibody, while exogenous Notch1-IC protein levels were visualised with an anti-Myc antibody by western blotting (top panel). The remaining extract fraction was tested for the occurrence of the indicated proteins (lower panel). We show a representative blot of three independent experiments