We have utilized integrated technologies including separation of proteins by 2-dimensional (2-D) gel electrophoresis and identification of proteins by matrix assisted laser desorption/ionizing time of flight (MALDI-TOF) mass spectrometry to examine an array of proteins that are regulated following treatment with neurotoxin. In essence, total cellular lysates harvested from MN9D dopaminergic neuronal cells treated with 6-hydroxydopamine (6-OHDA) for various time periods were subjected to 2-D gel separation followed by an analysis of the protein spots separated. Among the several protein spots that appeared to be either up- or down-regulated following 6-OHDA treatment, MALDI-TOF mass spectrometry revealed that an ER chaperone protein, calreticulin, was upregulated in a time-dependent manner. 6-OHDA-mediated up-regulation of this protein spot was reversed to the untreated control level when MN9D cells were co-treated with a pan-caspase inhibitor or an anti-oxidant. Immunoblot analysis using anti-calreticulin antibody confirmed this phenomenon. Since accumulation of altered proteins may be relevant in Parkinson's disease, our data suggest that regulation of chaperone activity in dopaminergic neurons comprises an additional cellular response to death-inducing stimuli.
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