![]() ![]() ![]() To characterize Parkin protein levels in mitochondria, fresh brain tissues from Hipk2 +/+ and Hipk2 −/− mice were homogenized and mitochondria were isolated using the Mitochondria Isolation Kit (Thermo Fisher Scientific). A, Western blot analyses detected Parkin protein levels in Hipk2 +/+ and Hipk2 −/− mouse substantia nigra and cerebral cortex. Loss of HIPK2 leads to elevated Parkin protein levels in Hipk2 −/− brain tissues, whereas HIPK2 overexpression promotes Parkin degradation via the proteasome pathway. ![]() Statistical analyses used a two-way ANOVA: * p < 0.05. Statistics used the Student's t test: ns, not significant * p < 0.05 ** p < 0.01 and **** p < 0.001. For each treatment paradigm, we set up one additional neuron culture without any neurotoxin treatment, which was considered as 0 concentration, and the percentage of survival in these cultures was counted as 100%. B, Quantification of TH + TuJ1 + or TH − TuJ1 + neurons from the ventral mesencephalon of E13.5 Hipk2 +/+ and Hipk2 −/− embryos showed that Hipk2 −/− TH + TuJ1 + and TH − TuJ1 + neurons were more resistant to MPP +-induced toxicity. The primary neurons, immunostained with anti-TH (green) and anti-TuJ1 (red) antibodies, were treated with DMSO (control) or MPP + (5 μ m) for 24 h before they were fixed and processed for image analyses. A, Immunofluorescent confocal microscopic images of primary neuron cultures using ventral mesencephalon of E13.5 Hipk2 +/+ and Hipk2 −/− embryos. Hipk2 −/− neurons are more resistant to mitochondrial toxins. ![]()
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