Figure 3.

Snapin is enriched in the cell bodies of developing hippocampal neurons and is found in all synaptosomal fractions. (A) Cultures of primary hippocampal neurons were grown for 7 and 12 d.i.v. and immunostained for snapin or cypin. (B) Snapin, cypin, and actin protein expression were assayed in 15 μg of extracts from neuronal cultures at different developmental time points by Western blotting. Snapin protein expression is low at 2 and 4 d.i.v. when primary dendrites are forming and branching, and expression increases by 7 d.i.v. when primary dendrites have stopped forming and higher order branches are forming. Snapin protein expression remains through 17 d.i.v., when spine formation begins. Cypin protein is expressed at all time points assessed, consistent with the idea that cypin plays a role in dendrite formation and branching. Actin expression serves as a loading control. (C) Snapin is found in all synaptosomal fractions, including synaptic plasma membranes (which are enriched in GluR1), the synaptic vesicle fraction (which is enriched in synaptophysin), and the synaptic cytosol. Like snapin, cypin is enriched in all synaptosomal fractions. Ten micrograms of each fraction was loaded.