1. Parkin message and protein are differentially affected by dopaminergic neuronal toxins.
Parkin message and protein are differentially affected by dopaminergic neuronal toxins. A, B, Differentiated PC12 cells were treated with 6-OHDA and MPP+ for the indicated times. Parkin mRNA levels were measured by qPCR (A) and normalized to α-tubulin levels. Parkin protein levels were analyzed by Western blotting (B). Results are representative of experiments that were performed three times; each condition was performed in triplicate in each experiment. *p < 0.01 compared with control (CTR) by Student's t test. C, Parkin protein levels were tested in detergent-soluble (S) and -insoluble (P) fractions of PC12 cells in response to 6-OHDA or MPP+. The blot is from a representative experiment, which was repeated three times. D, E, Primary cortical neuron cultures were treated with either 6-OHDA or MPP+ as indicated, and parkin mRNA and protein levels were tested by qPCR (D) and Western blotting (E). Each experiment was performed three independent times. The blot in E is from a representative experiment, and the densitometric quantification is an average of the three independent experiments. *p < 0.01 compared with control condition by Student's t test. F, Differentiated PC12 cells were treated with 100 μm 6-OHDA for 4 h, washed, and then incubated with the indicated inhibitors—ammonium chloride (NH4Cl), chloroquine (CQN), zVAD-fmk (zVAD), and MG132—for 4 h. Protein lysates were prepared and analyzed by Western blotting with the indicated antibodies. G, Differentiated PC12 cells were treated with 1 mm MPP+ for 16 h, washed, and then incubated with MG132 for 8 h. Samples analyzed as in F. H, Densitometric quantification of parkin protein levels after treatment with either 6-OHDA or MPP+, in either the presence or absence of MG132. *p < 0.05, **p < compared with toxin-treated condition without MG132 by Student's t test. The experiments with MG132 were performed three times with each toxin, with representative blots shown; the other inhibitors were used in one experiment.
Xiaotian Sun et al. J. Neurosci. 2013;33:
©2013 by Society for Neuroscience