Figure 9.

Trib3 overexpression decreases Parkin levels. A, B, Trib3 overexpression decreases Parkin protein levels in neuronal PC12 cells. Representative Western blot images (A) and corresponding quantifications (B) of Trib3 (top) and Parkin (bottom) protein levels in neuronal PC12 cells transduced with an empty control vector (pWPI empty) or a Trib3-expressing vector (pWPI Trib3) for the indicated times (1, 3, or 7 d). Protein levels were quantified and normalized to ERK protein levels, and values are mean ± SEM from five independent experiments. Multiple comparisons were performed using ANOVA with Student-Newman-Keuls post hoc tests: **p < 0.005; ***p < 0.0005. C, qPCR analyses of Trib3 (left) and Parkin (right) mRNA levels in neuronal PC12 cells, transduced with pWPI empty or pWPI Trib3 for 1–2 d, shows that Trib3 overexpression does not affect Parkin mRNA levels. Trib3 and Parkin mRNA levels are normalized against 18S rRNA, and values are mean ± SEM and were analyzed from three independent experiments performed in triplicate. ***p < 0.0005 (t test). D, E, Trib3 overexpression reduces Parkin expression in cultured postnatally derived ventral midbrain dopaminergic neurons. D, Representative immunofluorescence images of cultured postnatally derived ventral midbrain dopaminergic neurons immunostained for TH (blue), GFP (green, lentiviral-infected cells), and Parkin (red) after 2 d of infection with pWPI empty or pWPI Trib3. E, Quantification of relative Parkin immunostaining signals in dopaminergic neurons expressing pWPI Trib3 for 1–4 d, compared with dopaminergic neurons expressing pWPI-empty. ***p < 0.0005 (t test). Trib3 relative protein levels correspond to the mean ± SEM of Trib3 densitometric signals measured in 45–58 individual neurons and normalized to the corresponding control conditions. Values were analyzed from three independent experiments done in triplicate.