Figure 6. DYRK1A overexpression increases phosphorylation of p27^Kip1 and Cyclin D1 in SH-SY5Y cells and alters their protein levels. (A) Western blot analysis of total protein extracts from SH-SY5Y cells. Cells were treated with 2 µg/ml doxycycline to induce overexpression of DYRK1A or DYRK1A-K188R or with 10 µM RA. Control cells were left untreated (−). Whole-cell lysates were prepared 24 h or 72 h after induction of DYRK1A overexpression or differentiation and analyzed by immunoblotting with the indicated antibodies. Migration of mass standards is indicated in kDa (left). Densitometric evaluation of 3 independent experiments (means + SD) is shown in panels (B; Cyclin D1) and (C; p27^Kip1). (D) Overexpression of DYRK1A or DYRK1A-K188R was gradually induced using increasing concentrations of doxycyclin as indicated. Cells were lysed 24 h (left panel) or 72 h (right panel) after induction of DYRK1A overexpression, and whole-cell lysates were subjected to western blot analysis. Densitometric evaluation of 3 independent experiments (means + SEM) is summarized in panels (E) (24 h) and (F) (72 h). Ser10 and Thr286 phosphorylation were normalized to p27^Kip or Cyclin D1 total protein and p27^Kip or Cyclin D1 levels are shown relative to the loading control (GAPDH). One-sample t test was used to compare columns to normalized controls (B and C). One-way ANOVA + Bonferroni post-test was used to analyze effects of increasing dox concentrations. Significances are indicated for comparison of GFP-D1A with GFP-D1A-KR (F and G). p27^Kip1/GAPDH data (F) was tested using Kruskal–Wallis test and Dunn multiple comparison post-test; *P ≤ 0.05.; **P ≤ 0.01***P ≤ 0.001.