Figure 5.

PPM1B-mediated dephosphorylation of DYRK1A reduces tau phosphorylation at T212.A, HEK293 cells were transfected for 24 h with plasmid encoding V5-Tau, Myc-PPM1A, or Myc-PPM1B alone or in combination. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-V5 antibody. The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5 or anti-Myc antibodies. Hsp90 served as a loading control. B, HEK293 cells were transfected for 24 h with plasmid encoding V5-Tau, Xpress-DYRK1A, Myc-PPM1A, or Myc-PPM1B alone or in combination. Cell lysates were immunoblotted with anti-pTau antibody (T212). The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5, anti-Xpress, or anti-Myc antibodies. C, HEK293 cells were transfected for 24 h with plasmid V5-Tau, HA-DYRK1A-WT, HA-DYRK1A-S310A, HA-DYRK1A-4SA, or HA-DYRK1A-5SA alone or in combination. Cell lysates were immunoblotted with anti-pTau antibody (T212). The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5 or anti-HA antibodies. D, All graph data represent the mean ± SD of three independent experiments (∗∗∗p < 0.001; n.s., not significant). E, HEK293 cells were transfected for 24 h with plasmid encoding V5-Tau, HA-DYRK1A-WT, HA-DYRK1A-S258A, or HA-DYRK1A-S258D alone or in combination. Cell lysates were immunoblotted with anti-pTau antibody (T212). The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5 or anti-HA antibodies. The pThr212-tau/tau ratio is shown in (F). All graph data represent the mean ± SD of three independent experiments (∗∗∗p < 0.001). G, HEK293 cells were transfected for 24 h with plasmid encoding V5-Tau, HA-DYRK1A WT, HA-DYRK1A S258A, or Myc-PPM1B alone or in combination. Cell lysates were immunoblotted with anti-pTau antibody (T212). The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5, anti-HA, or anti-Myc antibodies. H, all graph data represent the mean ± SD of three independent experiments (∗∗∗p < 0.001; n.s., not significant). I, HEK293 cells were transfected for 24 h with plasmid encoding HA-DYRK1A-WT, HA-DYRK1A-S258A, or HA-DYRK1A-S258D. Cell lysates were immunoprecipitated with anti-HA antibody, and the samples were incubated for 30 min with kinase buffer, recombinant tau, and [γ-³²p]ATP. The reaction products were separated by SDS-PAGE and analyzed by autoradiography. Bacterial recombinant tau was stained with CBB. Proper expression of transiently expressed HA-tagged Dyrk1A-WT and its mutants was confirmed by immunoblotting with the anti-HA antibody. The ³²P-tau/tau ratio is shown in (J). All graph data represent the mean ± SD of three independent experiments (∗∗p < 0.01; ∗∗∗p < 0.001).