Fig. 4
The expression of the mutant of GAT-1(P361T) protein was reduced in non-neuronal cells and neurons. a-b. Mouse cortical neurons were transfected with the wildtype or the mutant GAT-1(P361T) cDNAs at day 7 in culture. The total lysates were harvested from mouse cortical neurons expressing the wildtype GAT-1YFP (wt) or mutant GAT-1(P361T)YFP transporters after 8 days of transfection (a). HeLa cells were transfected with the wildtype GAT-1YFP (wt) or mutant GAT-1(P361T)YFP transporters for 48 h (b). The total lysates were then analyzed by SDS-PAGE. Membranes were immunoblotted with rabbit anti-GAT-1 for both neuronal and HeLa cell lysates (1:200). In neurons, the protein band of endogenous GAT-1, at 67 KDa, was intense. The main protein bands run at 108 KDa in both the wildtype and the mutant conditions, representing the YFP-tagged GAT-1. c. The total protein integrated density values (IDVs) were measured. The abundance of the mutant GAT-1(P361T) transporter was normalized to the wildtype condition. In c, the total protein abundance was measured by adding up all the bands between 90 and 110 KDa. The total protein IDVs of either the wildtype or the mutant was normalized to its loading control. The abundance of the mutant transporter was then normalized to the wildtype. (*p < 0.05 vs wt in HeLa; **p < 0.01 vs. wt in Neuron, n = 4–5 different transfections)