Gt949

The Jump in T-Rex HEK293 (JumpIn)
overexpressing human EAAT2/3 were kindly provided by the RESOLUTE
consortium (http://re-solute.eu).^{21 (link)}l-glutamic acid monosodium
salt monohydrate, l-cysteine, and doxycycline hyclate were
purchased from Sigma-Aldrich (St. Louis, United States). (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]
aspartate (TFB-TBOA) was purchased from Axon Medchem (Groningen, The
Netherlands). GT949 was purchased from Tocris Bioscience (Bristol,
United Kingdom). xCELLigence PET E-plates 96 (ACEA Biosciences, San
Diego, CA, United States) were purchased from BioSPX (Abcoude, The
Netherlands). l-[3,4-³H]-glutamic acid (50.8 Ci/mmol)
was purchased from PerkinElmer (Groningen, The Netherlands). All other
chemicals were of analytical grade and were obtained from standard
commercial sources.

Purified SLC1A2 in gel filtration
buffer was incubated with 1 mM monosodium l-glutamate (Sigma)
with 0 nM, 10 nM, 1 μM, or 100 μM GT949 (Tocris) or 100
μM TFB-TBOA (Tocris) for a minimum of 15 min at room temperature.
The final protein concentration was 0.51 mg/mL. The sample was loaded
into Prometheus standard capillaries (NanoTemper) and the intrinsic
fluorescence at 350 and 330 nm was recorded by a Prometheus NT.48
(NanoTemper) as the samples were heated from 20 to 95 °C at 1
°C/min. The melting temperature (TM) was determined from the
inflection point of the first derivative of 350 to 330 nm fluorescence
ratio. Each biological replicate is a protein from an independent
purification. Three to six technical replicates were performed per
recording. TM of TFB-TBOA was used as an internal standard for protein
quality.