Figure 1

Characterization of synaptosome enrichment and localization of calpain-2 in the synapse. (A) Electron micrograph of synaptosome isolated from mouse brain cortex by sucrose density gradient. Synaptic vesicles in the presynapse (arrow head) and the post synaptic density; PSD (arrow) are marked. Scale bar = 200 nm or 0.2 µm (B) Enrichment of post synaptic density protein (PSD95) in synaptosomes. The synaptosome isolation was performed as described in Materials and Methods. Post nuclear supernatant (PNS) and synaptosomes (Syn; 8 μg of protein from each fraction/lane) were resolved by SDS-PAGE, and immunoblotted against antibodies to PSD95 and tubulin. Upper panel blots were probed for PSD95. Later, these blots were probed for tubulin (lower panel) without stripping. Densitometric analyses for PSD95 levels were normalized to tubulin. Unpaired two-tailed Mann-Whitney test was used. Results are represented as the mean ± SD (n = 6 mice). (C) Colocalization of calpain-2 with homer1 in cortex of WT and APP/PS1 mouse brain. Immunofluorescence staining indicates co-localization of calpain-2 (red) with homer1 (green) in both the cell body and neurites of the neurons. Arrows indicates the immunoreactivity of calpain-2 and homer1 in neurites. Calpain-2 and homer1 co-immunofluorescence was most prominent in neurites as shown in the higher fluorescence micrographs as insets. No staining was seen in mouse cerebral cortex section incubated with IgG control. Scale bar = 75 μm.