Figure 3

Immunoprecipitation of xDYRK1B. A, C-terminal sequences of human and Xenopus DYRK1A and DYRK1B. Amino acids identical with the immunogenic peptide used to raise the DYRK1B antibody are highlighted in bold. B, Immunoprecipitation of xDYRK1B. HeLa cells were transiently transfected with expression plasmids for xDYRK1B, GFP-DYRK1B or the empty vector (control). After immunoprecipitation with DYRK1B-specific antiserum (αD1B) or preimmune serum (Pre), bound proteins were subjected to Western blot analysis with a phosphotyrosine-specific antibody (P-Tyr) and the DYRK1B antiserum as indicated. Detection of IgG is shown as a loading control. The asterisk marks an unspecific band. C, Specificity of the DYRK1B antiserum. COS7 cells were transiently transfected with expression plasmids for the GFP fusion proteins of mammalian DYRK1A or DYRK1B as indicated on top of the lanes. Aliquots of the whole cell lysates (input) or proteins eluted from the immunoprecipitates (αD1B, Pre) were subjected to Western blot analysis with anti GFP and anti DYRK1B antibodies as indicated. Detection of IgG is shown as a loading control.