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Granule neurons from P6 rat cerebellum were cultured in vitro for 5 days in full medium (serum + KCl) and transfected with the indicated constructs along with cytomegalovirus (CMV)-β-Gal. Sixteen hours after transfection, cells were washed and placed in medium containing 25 mM KCl but no serum (Fig. 2, A to E), 5 mM KCl (Fig. 2F), or serum-containing medium (Fig. 2C). After 12 hours, cells were fixed and stained with an antibody to β-Gal and Hoechst dye. For kinetics studies, cells were fixed at various time points after transaction. Cells were scored in a blinded fashion as apoptotic or healthy based on their nucleic and cell body-neurite morphology.
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Supported by a NIH Mental Retardation Research Center grant (P30-HD18655), grants from NIH (NS28829 to M.E.G.) and Daiichi Pharmaceuticals, and an NIH training grant (5T32NS07112 to Z.M.). A.B. was supported by a postdoctoral research fellowship for physicians from the Howard Hughes Medical Institute. We thank E. N. Olson and J. D. Molkentin for providing the MEF2CR24L and MEF2CVP16 plasmids, J. Han and M. Zhao for the MEF2CS387A and GAL4MEF2C plasmids, and M. Wiedmann for technical help. We thank P. Dikkes for assistance in preparing rat brain sections and L. Hu for assistance with antibody preparation. We thank A. Shaywitz and M. Lin for their helpful review of this manuscript.
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