The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-)) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges.

Excitatory amino acid transporter 4 (EAAT4), a member of the high-affinity Na+/K+-dependent glutamate transporter family, is highly enriched in Purkinje cells of the cerebellum, although it is not restricted to these cells. The detailed expression of EAAT4 protein in different adult rat fore- and midbrain regions was examined. Despite moderate expression levels compared with the cerebellum, EAAT4 protein was omnipresent throughout the fore- and midbrain. With antibodies raised against the N-terminal mouse EAAT4 sequence, the highest protein expression levels were observed in the substantia nigra pars compacta, ventral tegmental area, paranigral nucleus, habenulo-interpeduncular system, supraoptic nucleus, lateral posterior thalamic nucleus, subiculum, and superficial layers of the superior colliculus. Relatively high levels of EAAT4 protein were also detected in the hippocampal principal cells, in the glutamatergic, gamma-aminobutyric acid (GABA)ergic, dopaminergic and most likely cholinergic cells of all nuclei of the basal ganglia, and in neurons of layers II/III and V of the cerebral cortex. The expression of EAAT4 was confirmed at the mRNA level in some important fore- and midbrain structures by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) and estimated to range from 6.7 to 1.6% of the amount in the cerebellum as measured by real-time PCR.