Synaptic localization of rEag1 channels. (A) Hippocampal neurons were double-stained for rEag1/rEag2 (left panels) and the postsynaptic density marker PSD-95 (middle panels). Scale bar, 25 μm. (B) Quantification of the number of puncta per 100-μm neurite (puncta/100 μm) for PSD-95, rEag1, and rEag2. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from PSD-95. Data were collected from 7-11 different neurons. (C) Quantification of the co-localization of PSD-95 with rEag1 or rEag2. The data illustrate the fraction of PSD-95 puncta that were co-localized with rEag1/2 puncta, as well as the fraction of rEag1/2 puncta that were co-localized with PSD-95 puncta. The number in parenthesis denotes the amount of neurites analyzed, and the asterisk indicates a significant difference (t-test, p < 0.05) from rEag1. Data were collected from 7-8 different neurons. (D) Subcellular fractionation of rat brains: the homogenate (H), the soluble fraction (S1), the crude membrane fraction (P2), the synaptosomal fraction (SPM), and the two postsynaptic density (PSD) preparations (PSD I: one Triton X-100 wash; PSD II: two Triton X-100 washes). The left panel (25 μg) illustrates the primary fractionation profile, whereas the right panel (5 μg) exemplifies the further enrichment pattern in the three sub-fractions of synaptosomes. All fractions were subject to immunoblotting analyses with the indicated antibodies. 25 μg and 5 μg refer to the amount of total protein loaded in each lane.