Fig 2: The phox domain of Nischarin interacts with GLT-1(A) Schematic diagram depicting full length Nischarin and GFP-tagged mutants. GLT-1aV5 coimmunoprecipitated with GFP-phox but not GFP-Nisch?phox mutant or GFP control.(B) Coimmunoprecipitation experiments from mouse brain homogenate from WT and GLTKO mice, showing Nischarin to be part of a native complex with GLT-1 (n = 3).(C) Schematic diagram of GST fusion constructs for GLT-1 N-terminus, 15 amino acid stretches of GLT-1 N-terminus (A-E), GLT-1 C-terminus, and GLAST C-terminus. GFP-Nisch was successfully pulled down with full-length GST-fused GLT-1 N-terminus and to GST fusions D (amino acids 9-23) and E (amino acids 23-37).

Fig 3: Schematic of cell separation techniques.A) Positive selection of astrocytes was performed using an ACSA-2 antibody. Dissociated cells were incubated with magnetic beads conjugated with an ACSA-2 antibody, and were applied to a ferromagnetic column placed in a magnetic field. The astrocyte enriched cell population was eluted from the column after successive washes that removed non-target cell populations. B) Neurons were enriched using a negative selection protocol. Dissociated cells were incubated with biotin-conjugated antibodies targeting non-neuronal cell populations, followed by labelling with streptavidin-coated magnetic beads. Neuronal cell populations were eluted, while biotin-bound cells were retained on the magnetic column. C) Positive selection of cells is customizable. Here an antibody which detects an extracellular epitope of GLT-1 was used to enrich astrocytes. Dissociated cells were labeled with the rabbit-anti-GLT-1 antibody and subsequently labeled with anti-rabbit microbeads. The enriched population was retained on the column, and eluted subsequent to washing undesired cells from the magnetic column.

Fig 4: Nischarin mediates glutamate-dependent GLT-1 internalization in astrocytes(A) Surface biotinylation assay showing surface GLT-1 level in astrocytes transfected with GFP and GLT-1a-V5 or GFP-Nisch and GLT-1a-V5 following +/- 100µM glutamate treatment. One-way ANOVA, post hoc Dunnett’s multiple comparison test (n = 4 individual experiments).(B) Proximity ligation assay in DIV14 hippocampal culture. Increased red puncta per nuclei (DAPI stained (blue)) is indicative of increased direct interaction between Nischarin and GLT-1 in hippocampal culture. Glutamate treatment (100µM, 1h) significantly increased GLT-1-Nischarin interaction compared to control. One-way ANOVA, post hoc Tukey’s test (n = 3 individual preparations).(C) Schematic representation of GLT-1BBS construct bound to BTX conjugated Alexa 555 (BTX555). Astrocytes expressing GFP-Nisch and GLT-1aBBS were labeled using BTX555 and dual color live-structured illumination microscopy-monitored trafficking of GLT-1 following glutamate treatment. Merged kymographs of GFP-Nisch vesicle (green) and GLT-1 bound BTX-555 (red) reveal co-localized diagonal trajectory, representing moving vesicles.(D) Quantification of GFP-Nisch and GLT-1aBBS expressing astrocytes treated with 100µM glutamate for 0, 5, 30, and 60min showed increased colocalization between Nisch and GLT-1 compared to untreated controls. p values by unpaired t test, Mann Whitney test (n = 6-14).

Fig 5: Antibody feeding assay revealed that Nischarin promotes internalization of GLT-1(A–C). Surface and internal GLT-1 populations labeled in HeLa cells co-expressing (A) GFP and GLT-1a-HA or (B) GFP-Nisch and GLT-1a-HA at 0 min and 60 min. (C) Quantification of internalized GLT-1 to total GLT-1 levels at T60 min relative to T0 min. One-way ANOVA, Kruskal-Wallis test with Dunn’s correction (n = 12).