Table 1.
Details of antibodies used for fluorescent immunohistochemistry and Proximity Ligation Assay.
Figure 1.
2D gel separation and blotting with SLC38A6 antibody of the total protein in mouse brain.
A) Insoluble fraction of total protein indicating presence of SLC38A6 with the specific band between 37 and 50 KD. B) Soluble fraction of total protein indicating absence of SLC38A6 with no bands. No unspecific bands were detected in any of the fractions, validating the specificity of the antibody.
Figure 2.
Fluorescence micrograph of immuno-labelling on paraffin-embedded mouse brain tissues (7 mm thickness) with 40X and 10X magnifications, respectively.
In all panels, the nucleus is stained with DAPI in blue: A) Co-localization of SLC38A6 and NeuN is demonstrated with 40X magnification, where SLC38A6 is detected in green and the neural cells are stained in red. B) Co-localization between SLC38A6 and NeuN is further demonstrated with 10X magnification. C) No co-localization between SLC38A6 and glial cells/astrocytes is observed. SLC38A6 is detected at 40X magnification in green and glial cells are stained in red. D) No overlap between SLC38A6 and GFAP was recorded at 10X magnification. E) Expression of SLC38A6 in glutamatergic interneurons is illustrated at 40X magnification where SLC38A6 is detected in green and PAG is labeled in red. F) The overlap between SLC38A6 and PAG is demonstrated at 10X magnification.
Figure 3.
Confocal micrograph (63 times magnification) and PLA data confirming significantly higher expression of SLC38A6 in the neurons compared to the glial cells on paraffin-embedded tissue sections of whole mouse brain.
A) Three panels illustrate SLC38A6 staining in green (DAPI in blue), NeuN in red (DAPI in blue) and three color overlay (merged), respectively. B) Three panels here represent SLC38A6 staining in green (DAPI in blue), GFAP in red (DAPI in blue) and three color overlay (merged), respectively. C) Quantification of PLA signals per cell exhibits higher interaction between SLC38A6 and NeuN (neuronal marker) than that between the transporter and GFAP (glial cell marker), confirming no or minor expression of SLC38A6 in the glial cells compared in the neurons. Statistical analysis (t test) shows that interaction between SLC38A6 and NeuN is significantly higher than that between SLC38A6 and GFAP.
Figure 4.
Fluorescence micrograph of immunohistochemistry executed on primary cell culture of mouse embryo cortex at 20X magnification.
A) VIAAT or VGAT protein tagged with eGFP is viewed in green and the nucleus is stained with DAPI in blue. B) SLC38A6 protein is visualized with Alexa-flour 594 (AF594) secondary antibody in 1∶400 dilution in red and the nucleus is stained with DAPI in blue. C) Merged image of VIAAT/VGAT-eGFP in green and SLC38A6-AF594 in red together with the nucleus, stained with DAPI in blue. D) Quantification of VIAAT/VGAT positive cells and SLC38A6-expressing cells and their overlap in comparison with total number of cells in each tissue section. The first column exhibits total number of cells, whereas the second and third column represents VIAAT/VGAT positive (green signals) and SLC38A6-expressing (red signals) cells, respectively. The last column shows overlap between green and red signals, representing the cells expressing both VIAAT/VGAT and SLC38A6 proteins. The number of cells counted was 1193 (in total). Statistical analysis (1way ANOVA) exhibits significantly higher number of cells expressing SLC38A6 compared to cells with overlapping red and green signals.
Figure 5.
Fluorescence micrograph of N25/2 hypothalamic cell line with 40X magnification.
Nucleus is stained blue with DAPI in all panels. A) Simultaneous localization of SLC38A6 (in green) and WGA (in red) is visualized. B) Localization of SLC38A6 (in green) in context with synaptotagmin (in red) is demonstrated here. C) Localization of SLC38A6 (in green) in relation with Snap25 (in red) is illustrated.
Figure 6.
Proximity ligation assay (PLA) on N25/2 hypothalamic cell line to demonstrate protein-protein interaction pattern between SLC38A6 and neuronal markers.
The nuclei of the cells are stained in blue with DAPI. The PLA signals are detected with red filter. A) SLC38A6 interacts with Snap25 exhibiting proximity to the membrane. B) SLC38A6 interacts with Synaptotagmin, illustrating substantial proximity to the vesicular proteins. C) PLA control with no primary antibody incubation and hence no signal of interaction between proteins demonstrating successful PLA in the treated cells. D) Signals per cell were counted and represented here as histograms. Columns 1, 2, 3, 4 and 5 reveal interactions between Synaptotagmin-Synaptotagmin, Snap25-Snap25, Synaptotagmin-Snap25, SLC38A6-Synaptotagmin and SLC38A6-Snap25, respectively. Columns 1 and 2 exhibit quantification of PLA signals obtained by using different antibodies on the same proteins, Synaptotagmin and Snap25 respectively. Column 3 depicts interaction between synaptotagmin and Snap25 to illustrate their proximity in the cells. Columns 4 and 5 show interactions between SLC38A6 and synaptotagmin and Snap25, respectively. E) Normalized signals per cell are shown here to compare interactions of SLC38A6 with Synaptotagmin and Snap25, respectively. Column 1 and column 2 demonstrate normalized interactions between SLC38A6 and synaptotagmin and SLC38A6 and Snap25, respectively.
Figure 7.
Schematic diagram of relative location of SLC38A6 in the cell.
According to our proposed model, SLC38A6 is located at the membrane of synapse and is shown as a trans-membrane protein in dark blue. It interacts with the well-known membrane protein Snap25 present at the synaptic junction/cleft and is represented here as light blue circles. The vesicular protein Synaptotagmin is shown in green and is interacting with SLC38A6 when the vesicles are in close proximity to the membrane, prior to the release of neurotransmitters. Some of the relevant interactions, as recorded by PLA signals, are shown by red double arrows.