Comparative analysis of antibodies to xCT (Slc7a11): Forewarned is forearmed

ABSTRACT 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. J. Comp. Neurol. 524:1015–1032, 2016. © 2015 Wiley Periodicals, Inc. Using xCT knockout brain (xCT−/−) as negative control, we show that antibodies specifically recognizing xCT, the specific system Xc− subunit, label a protein with a molecular weight of 35 kDa (A), whereas many commercially available antibodies detect a 45–50‐kDa band (B). Only one in‐house‐developed antibody can be used for immunohistochemistry (C,D)