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. 2016 May 9;7(26):39184–39196. doi: 10.18632/oncotarget.9258

Figure 3. Localization of Prrt2 at the pre- and post-synaptic membranes.

Figure 3

A. Membrane fractionation assay and post-synaptic density (PSD) preparation of the protein from an adult rat brain. Prrt2 and SNAP25 were present and co-fractionated in both pre- and post-synaptic compartments. PSD95 and GluA1 are PSD proteins that were enriched in PSD fractions. Synaptotagmin I (Sygt1)-a presynaptic vesicle protein was present in the LP2 fraction, but absent from all PSD fractions. PSD95, GluA2 and N-cadherin are post-synaptic membrane proteins that were absent from the LP2 fraction. H = homogenate, S1 = low-speed supernatant, P1 = nuclear pellet and debris, S2 = cytosol plus light membrane, P2 = crude synaptosomal fraction, S3 = cytosol, P3 = light membranes, LS1 = synaptosomal cytosolic fraction, LP1 = synaptosomal membrane fraction, LS2 = luminal fraction of synaptic vesicles, LP2 = synaptic vesicle membrane fraction. PSDI, II and III represent the detergent extractability of PSD resident proteins. PSD1 represents Single Triton X-100 extraction. PSDII and PSDIII were obtained in the second round of Triton extraction and Sarkosyl extraction of PSDI, respectively. B. Immunostaining of Prrt2 and SNAP25 in GFP-expressing cultured hippocampal neurons (DIV 17). A dendritic segment with multiple spines (specialized protrusive structures) is shown in the large image. The amplified images highlight the localization of Prrt2 (red) and SNAP25 (blue), which co-localized at the tip of a spine (orange arrow) where a synapse was forming.