not-yet-known not-yet-known not-yet-known unknown [3H]GABA uptake Acute hippocampal slices were used for GABA uptake experiments as previously (Chazalon et al., 2018) with modifications. After the recovery period (at least 1 hour), slices were transferred to a 24-well plate (Corning Costar, Corning, NY). Three slices were placed in each well (for triplicate measurements per condition per experiment) containing 1.2mL non-supplemented low-glucose DMEM (Gibco™ 11880028, Invitrogen) and the appropriate drug concentrations; incubation was for 30 minutes under oxygenation (5% CO2, 95% O2) at room temperature. Slices were then gently transferred to a prefilled and prewarmed (37ºC under an oxygenated atmosphere) 48-well plate (300 μL and 1 slice per well) for incubation with 25μM [3H]GABA (Revvity/PerkinElmer, specific activity in stock: 25–40 µCi/nmol; in the incubation solution due to addition of cold GABA: approx. 22 nCi/nmol) plus the same drugs as before. Zero time for incubation with [3H]GABA was taken as the time of slice transfer. [3H]GABA uptake was allowed for 12 minutes (to allow equilibrium) and stopped by a quick wash in ice-cold aCSF solution (approx. 10 mL in a Petri dish) followed by immediate transfer into 200 µL ice-cold lysis buffer solution. Each slice was then homogenized for posterior quantification of tritium (liquid scintillation counting) and protein (detergent compatible protein assay kit, Bio-Rad, California, USA) content in each sample. In each step, slices were transferred sequentially at defined time intervals to allow proper incubation times for each slice. To minimize time-related bias, a complete sequence of drug conditions was run first, then (and by the same order) the duplicates were run, and then the triplicates. For each test drug condition, GABA uptake was assessed in consecutive wells without or with the GAT-1 selective blocker, SKF89976A (20 μM). GAT-1-mediated uptake was calculated by subtracting the amount of tritium in the samples incubated in the presence of the GAT-1 blocker (non-GAT-1 mediated) from the total tritium detected in the samples incubated in the same conditions but in the absence of the GAT-1 blocker. 0.1 Western Blotting Human hippocampal samples were lysed in 200 µL of radioimmunoprecipitation assay (RIPA) lysis buffer (4% nonyl phenoxypolyethoxylethanol (NP-40), 40 mM Tris-HCl, 1 mM ethylenediamine tetraacetic acid (EDTA), 150 mM NaCl, and 0.1% sodium dodecyl sulphate (SDS) 10%), 10 mM NaF, 5 mM Na3VO4 and a protease inhibitor (cOmpleteTM Mini EDTA-free Protease Inhibitor Cocktail: Roche, Basel, Schweiz). Following homogenization with a sonicator (Soniprep MSS 150.CX3.5: Sanyo, Osaka, Japan), total protein was quantified (DC Protein Assay Kit, Bio-Rad, California, USA), and absorbance was measured at 750 nm in the Infinite M200 (Tecan Trading AG, Hombrechtikon, Switzerland). Lysates were denatured for 10 minutes at 95 °C in sample buffer (12% SDS, 0.06% bromophenol blue, 47% glycerol, 600 mM dithiothreitol, 60 mM Tris-HCl pH 6.8). Each sample (40 µg total protein/well) and molecular weight marker (MWM, NZYColour Protein Marker II: Nzytech, Lisboa, Portugal) were separated on 10% SDS-polyacrylamide (SDS-PAGE) gel within a running buffer (25 mM Tris-base, 192 mM glycine, 10 mL SDS 10%, dissolved in Mili-Q® water, pH 8.3) at 80 volts, until the marker started to separate and then, at 120 volts for approximately 90 minutes using Mini-Protean® Tetra System (Bio-Rad, Hercules, California, USA). SDS-PAGE-separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, California, USA) inside a transfer buffer (25 mM Tris-base, 192 mM glycine, 10% methanol, dissolved in Mili-Q® water, pH 8.3) using the same system at 250 mA for 1 hour and 45 minutes. Membranes were blocked for 1 hour at RT in 3% Bovine Serum Albumin (BSA) in TBS-T (20 mM Tris-base, 137 mM NaCl, 0.1% Tween-20, pH 7.6). Subsequently, membranes were incubated with the primary antibodies − Adenosine A3 Receptor (A3R) rabbit antibody (AA 85-180, 1:1000, antibodies-online GmbH, Aachen, Germany) overnight at 4 °C, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (AM4300, 1:5000, Invitrogen, Massachusetts, USA) for 40 minutes at RT − followed by 1 hour at RT with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG-HRP, 1706515, 1:5000, Bio-Rad; Goat anti-Mouse IgG-HRP, 1706516, 1:5 000, Bio-Rad). All antibodies were diluted in TBS-T with 3% BSA. In between steps, membranes were rinsed in TBS-T (3 times for 5 minutes). Immunoreactions were detected with Western Lighting Pro ECL (PerkinElmer Inc, Massachusetts, USA), which generates a sensitive light-emitting product through HRP action, and chemiluminescence was detected using an Amersham ImageQuant 800 system (Cytvia, Marlborough, USA). Fiji software (National Institutes of Health, Maryland, USA) was used to quantify band intensities while maintaining the same area between bands within the same membrane. The intensity of each protein band was normalised to that of GAPDH, and then to the mean of controls on the same membrane. 0.1 Western Blotting