Analysis of Cultured BV-2 Cell Proteome in Response to Select •NO Producing Activators by Western Blot, Two Dimensional Gel Electrophoresis and MALDI TOF/TOF
Sherman, Rachel F.
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Activated microglia have been found to be intimately associated with Ap-plaques in the brains of Alzheimerfs patients. The exact relationship between nitric oxide (•NO, released by activated microglia) and the senile plaques is not currently well understood. In an attempt to elucidate the mechanisms involved in activation of these cells, the effects of different activators were examined in BV-2 cells. Western analysis showed that the levels of •NO release (measured as nitrite) following treatment with phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), y-interferon (y-INF), or a combination ofthese, corresponded for the most part to levels of iNOS protein expression. The role ofPKC in the regulation of •NO release was also examined, and it was found that inhibition of the a, p, y, and [i PKC isoforms completely eliminated •NO production in all cell treatments except for in cells treated with a combination LPS/y-INF; only 50% ofthe activity was inhibited. Protein profiling ofthe BV-2 proteome was also done on cell lysates after treatment with the cell activators. Select altered proteins, as analyzed by two-dimensional electrophoresis, were identified by matrix-assisted laser desorption/ionization-time offlight mass spectrometry (MALDI-TOF MS) following tryptic digestion. Nine ofthe ten selected proteins were positively identified by this technique as being different proteins: calreticulin, ATP synthase, vimentin, chaperonin, actin, a-enolase, gag protein, cofilin 1 and phosphoglycerate mutase. These proteins have been implicated in regulating cytoskeletal structure, redox status, signal transduction, glycolysis, transcription and protein folding. Possible connections between effected proteins and the progression of Alzheimer's disease will be made.