(a-c) Size-exclusion chromatograms of purified EAAT1CO (a), EAAT1COCO (b), and the chimeric transporter EAAT1-ScaDCO-TranDCOCO (c), respectively, pre-heated at different temperatures. The chromatograms at all temperatures were normalized to the peak fluorescence value observed at 4°C, and show how the trimeric form of the transporters that elutes at ~3.0 ml unfolds into lower oligomeric state(s), most likely monomers that elute at ~3.5 ml. (d-f) Thermal unfolding curves depicting the change in fractional area of the chromatographic peak corresponding to the trimeric transporters (black symbols), as a function of the pre-pulse temperature in EAAT1CO (d), EAAT1COCO (e), and EAAT1-ScaDCO-TranDCOCO (f), respectively. Solid lines indicate fits of a Hill-like equation (see methods) to the data with T50-SEC values 49.06 ± 0.1, 38.4 ± 0.4°C and 45.6 ± 0.4°C, and H values −21.0 ± 0.3,–9.3 ± 0.7, and −18.3 ± 5.3 for EAAT1CO, EAAT1COCO, and EAAT1-ScaDCO-TranDCOCO, respectively. The total area under the chromatogram at each temperature, normalized to that at 4°C, is also shown (empty symbols), and remains relatively constant at all temperatures showing the lack of protein aggregation or loss during sample preparation. Plots in (d), (e), and (f) depict an average of at least three independent experiments (circles), and error bars represent s.e.m.