The Journal of Neuroscience, October 11, 2006, ():
Cystine/Glutamate Exchange Modulates Glutathione Supply for Neuroprotection from Oxidative Stress and Cell Proliferation
J. Neurosci. Shih et al.
26: 10514
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure 1. Verification of xCT band migration using HA-tagged xCT and Nrf2 knockout tissues. A, To verify xCT molecular weight in Western blot, HA-tagged xCT was expressed in HEK293 cells and cell lysates were probed with anti-HA and anti-4F2hc antibodies. Monomeric HA-xCT was observed as a 35 kDa and 55 kDa band, consistent with the peptide-derived xCT antibody. The 105 kDa band corresponding to the HA-xCT-4F2hc heterodimer could be disrupted under reducing conditions (β-ME + dithiothreitol), as observed with both HA and 4F2hc antibodies. The 55 kDa HA-xCT band referred to as xCT-mod could not be disrupted by reducing conditions suggesting that it is not a di-sulfide linked xCT homodimer. Blots are representative results from three independent experiments on separately prepared cultures. B, The peptide-derived xCT antibody was also used to probe brain tissues from Nrf2 wild-type and knockout mice. Consistent with the xCT-specific action of our antibody, we observed a significant reduction of xCT protein expression in meninges, and a modest reduction in cortex of Nrf2 knockout mice, compares to wild-type littermates. However, overall basal levels of xCT expression throughout other brain regions were largely unaffected by Nrf2 knockout. Hippo. = hippocampus, cerebell. = cerebellum. Data represent the mean + SEM from n = 3 separate mice from each genotype. * p < 0.05, compared to Nrf2+/+ mice.
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Supplementary Figure 2. Total regional brain GSH levels in the embryonic and adult rat brain. A,B, Brain homogenates were collected from both age groups and assayed in the same experiment using the Tietze method (Tietze, 1969). GSH levels in embryonic brain were ~ 5x higher in all regions (note scale bars). Data represent the mean + SEM from n = 3 rats for each age group.
