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. 2022 Jun 6;4(3):fcac144. doi: 10.1093/braincomms/fcac144

Figure 3.

Figure 3

4-Phenylbutyric acid (PBA) concentration and time dependently increased the GABA uptake in cells expressing the wildtype or the mutant GAT-1. (A, B) HEK293T cells were transfected with wildtype GAT-1YFP (wt) for 48 h, PBA (2 mM) was applied dropwise to each dish at different concentrations (A) and incubated for time durations (B). PBA from stocking solution (2 M) was diluted with DMEM 100 µL to desired concentration. (A) The graphs represent the altered GABA reuptake function of the wildtype GAT-1YFP in HEK293T cells treated with PBA for a series of different concentrations. The GABA uptake activity of cells treated with PBA of different concentrations was normalized to the sister cultures treated with DMSO alone for 24 h. (B) The graphs represent the altered GABA reuptake function of the wildtype GAT-1 in HEK293T cells treated with PBA for a series of different time duration over treated with DMSO alone for 24 h. GABA uptake activity was measured by the high-throughput 3H radio-labelling GABA uptake on a liquid scintillator with QuantaSmart. (C) Cartoon showing the mutant allele only was expressed. HEK293T cells were transfected with the wildtype or the mutant GAT-1YFP cDNAs alone for 48 h. (D) Cartoon showing the coexistence condition of the wildtype and the mutant allele in patients and both the wildtype and the mutant alleles were expressed. HEK293T cells were transfected with the wildtype GAT-1YFP alone or in mixture of the wildtype or the mutant cDNAs for 48 h. In the mixed condition, the ratio of the wildtype GAT-1 with pcDNA or the mutant cDNAs are 1:1 with the total cDNA amount of 0.5 µg. PBA (2 mM) was applied for 24 h, while DMSO was applied as control. Both wt and the mutant were normalized to its own DMSO-treated conditions. Around 966 stands for the wildtype treated with Cl-966 (50 µM), while 711 stands for NNC-711 (35 µM). N = 4–7 different transfections. In A and B, *P < 0.05, **P < 0.01, ***P < 0.001 versus wt 0. In C and D, *P < 0.05, **P < 0.01, ***P < 0.001 versus its own DMSO treated. In C, ns stands for no significance. One-way analysis of variance and Newman–Keuls test. Values were expressed as mean ± SEM.