Rationale: The proline transporter (PROT) is an integral membrane protein expressed in a subset of excitatory (glutamatergic, asymmetric) synapses in different brain regions.
Pre- and postembedding immuno-EM reveals a localization of PROT predominately to synaptic vesicles in a subset of glutamatergic terminals, but also to the plasma membrane (Fig. 5B).
Antibodies: Polyclonal rabbit antibody A2 was produced and affinity-purified by Research Genetics (Huntsville, AL) against residues 2–18 from the predicted N terminus of rat PROT (KKLQEAHLRKPVTPDLL). The affinity-purified polyclonal anti- body directed against the C terminus of rat PROT, antibody C597, has been described previously (Velaz-Faircloth et al., 1995). Both antibodies were tested for its specificity by biochemistry using stably PROT expressing HEK cells and transiently expressing HeLa cells (Fig.1). C597 antibody, preabsorbed with the C-terminal antigenic peptide, did not produce any immunoreactivity by immunohistochemistry (Fig.2D).. Experimental description: For electron microscopy, four adult (250–350 gm) male Sprague Daw- ley rats (Hilltop Lab) were anesthetized...
Subcellular fractionation: Rat brain synaptic vesicles were purified from isolated nerve terminals...