Figure 2

D-[³H]-Asp uptake activity and membrane expression of mutants.

HeLa cells were transfected with the CL-GLT-1 or constructed mutants. (A) Transport activity of the mutants were measured in Hela cells as described under Materials and Methods and expressed as a percentage of CL-GLT-1. (B) The total proteins, biotinylated membrane proteins and non-biotinylated proteins were measured by western blot as described under Materials and Methods. Blots of all proteins were probed with the anti-GLT-1 antibody. Each blot of the biotinylated membrane proteins was probed for the internal plasma membrane marker -integrin and the absence of α/β-tubulin, an endogenous cytosolic protein representing the negative control in the biotinylated membrane proteins. Each blot of the non-biotinylated proteins was probed for the presence of α/β-tubulin. (C) Densitometric analysis of the biotinylated membrane proteins for each mutant normalized to internal marker (integrin) and represented as a percentage of CL-GLT-1. (D) D-[³H]-Asp uptake activity normalized to relative cell surface expression. (E) The ratio of the biotinylated membrane protein expression and non-biotinylated protein expression of CL-GLT-1 and mutants. Values represent the means ± S.E. of at least three different experiments done in triplicates. Values that are significantly different from that of CL-GLT-1 were determined by one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.01).