Figure 5

Protein localization and interactions of B⁰AT3 to glutamatergic neurons and vesicles. Immunohistochemistry (A-C) on mouse brain and spinal cord and proximity ligation assay (PLA) (D-E) on a hypothalamic mouse cell line are illustrated here with B⁰AT3 immunoreactivity in red, markers in green, and nucleus marker DAPI in blue. (A) Highly overlapping expression in glutamatergic neurons between B⁰AT3 and the enzyme PAG in cerebral cortex in the brain (Bregma 1.18). (B) Low overlapping expression for the vesicular marker VGLUT1 and B⁰AT3 in dorsal horn in spinal cord (upper vertebrae L2 lumbar). (C) Expression of the vesicular glutamate transporter VGLUT2 extensively overlapped with the expression of the B⁰AT3 transporter in ventral horn in spinal cord (Bregma −0.10). Fluorescent micrographs of paraffin sections taken with 40x magnification. PLA signals (red fluorescent amplification product) and the nuclei (blue) are shown as an increased intensity projection of the raw image based on 20 z-planes (D-F). (D) No interactions were seen in the negative control that was run without primary antibodies. (E) Moderate number of interactions detected between B⁰AT3 and synaptophysin. (F) High number of interactions detected between B⁰AT3 and PAG (scale bar, 10 μm).