Dyrk1B protein expression and turn-over in Neuro 2a cells.

<p>(<i>a</i>) Dyrk1B expression in transiently transfected Neuro 2a cells was examined at 16 h and 48 h post-transfection in the presence or in the absence of the specific proteasome inhibitor MG132. Dyrk1B is not detectable in non-transfected Neuro 2a cells (CTL) with available antibody, while transgene Dyrk1B, obvious at 16h, is decreased overtime in a proteasome-specific manner (upper panel). In the absence of MG132, cyclin D1 levels are inversely related to Dyrk1B (middle panel) while in the presence of MG132 cyclin D1, also degraded via the proteasome, is maintained despite persisting Dyrk1B expression (middle panel); β-tubulin indicates protein loading (lower panel). (<i>b</i>) Dyrk1B is localized in the nucleus of Dyrk1B⁺ Neuro 2a cells 16 h post-transfection, while at 48 h it is mainly cytoplasmic as shown by immunocytochemistry and confocal microscopy. The cells depicted by arrows at 16h and 48h, respectively, are shown at higher magnification. Different image acquisition settings were used at 16h and 24h to compensate for signal reduction at 48h. Scale bar: 10 μm. (<i>c</i>) Dyrk1B protein turn-over in Neuro 2A cells. Cells were transfected with Dyrk1B and allowed for expression 16 h after transfection. Cells were then treated with cycloheximide for different times as indicated and subjected to Western blot analysis. (<i>d</i>) Dyrk1B reduces BrdU incorporation in transiently transfected Neuro 2a cells after 16 h of expression. **Student’s t-test, p=0.00146, n=5. (<i>e</i>) In the presence of 10 μM harmine, a specific kinase inhibitor of the Dyrk1 protein family, the Dyrk1B-dependent down-regulation of cyclin D1 is inhibited in Neuro 2a cells (middle panel) without affecting Dyrk1B protein levels (upper panel). Lanes show Neuro 2a cells transiently transfected with Dyrk1B and allowed 16 h for expression, or non-transfected cells (CTL), harvested and immunoblotted with the indicated antibodies. (<i>f</i>) Quantification of Dyrk1B protein levels normalized relative to β-actin, in the presence or absence of 10 μΜ harmine. (<i>g</i>) Cyclin D1 is wiped out from the nuclei of Dyrk1B⁺ transiently transfected Neuro 2a cells (arrows, <i>i</i>-iv) but is clearly maintained in the nucleus of Dyrk1B⁺ cells in the presence of harmine (arrows, <i>v</i>-viii). Cells were double labeled for Dyrk1B (green) and cyclin D1 (red) while nuclei were visualized using TO-PRO-3 (blue). The merged pictures are shown (iv, viii). Scale bar: 8 μm.