System xc−-specific 14C-L-cystine (PerkinElmer; Waltham, MA) and system XAG−-mediated 3H-D-aspartate (PerkinElmer; Waltham, MA) uptake was performed as previously described (Fogal et al., 2007 (link)). Cultures were washed into HCSS (3 × 750 µl) and allowed to equilibrate for 10 min (25℃). For cystine uptake, cells were incubated in HCSS containing 3 µM 14C-L-cystine (1 µCi/ml), 27 µM unlabeled cystine, 1 mM D-aspartate, and 0.5 mM acivicin (Enzo Life Sciences; Farmingdale, NY). D-aspartate and acivicin were included in the uptake buffer to block system XAG− and γ-glutamyltranspeptidase, respectively. Uptake was terminated after 30 min by washing in ice-cold PBS (3 × 750 µl). For D-aspartate uptake, cells were incubated in HCSS containing 0.1 µCi/ml 3H-D-aspartate and 50 µM unlabeled D-aspartate (25℃) for 5 min and uptake terminated by washing cells with an ice-cold Na+-free choline stop buffer containing in mM: 116 choline chloride, 0.8 MgSO4, 1 KH2PO4, 10 HEPES, 5 KOH, 10 glucose, 0.9 CaCl2, and 5 nonradioactive D-aspartate.
Cells were lysed with warm 0.5% SDS and accumulated radioactivity estimated using a liquid scintillation counter. Readings of counts per minute from experimental conditions were corrected back to the original volume of lysate, and the picomoles of cystine and aspartate transported per minute were calculated as described (Fogal et al., 2007 (link)).
Cells were lysed with warm 0.5% SDS and accumulated radioactivity estimated using a liquid scintillation counter. Readings of counts per minute from experimental conditions were corrected back to the original volume of lysate, and the picomoles of cystine and aspartate transported per minute were calculated as described (Fogal et al., 2007 (link)).