Figure 2: Transsulfuration is a significant source of sulfur for glutathione synthesis in human mammary cells. human mammary epithelial cells (HMEC’s) were pretreated with vehicle control, pathway inhibitors buthionine sulfoximine (BSO), or propargylglycine (PPG) for 24 hours then labeled with 35S-methionine for 24 hours. Lysate and glutathione bimane conjugates were prepared and analyzed by thin layer chromatography and autoradiography as described under methods. Comparison of the images (fluorescence lanes 1 and 2 with autoradiography lanes 1 and 2) indicates that the fluorescent GSH-MCBi conjugate is radioactive. Incorporation of  35S-methionine into glutathione (GSH-MCBi bands) demonstrates that functional transsulfuration occurs in mammary cells. PPG inhibitory impact on either glutathione synthesis from all cysteine sources (left panel, fluorescence, compare PPG with control) or the incorporation of  35S-methionine labeled cysteine (which must be transsulfuration derived) into glutathione demonstrates both transsulfuration and the identity of the TLC spots. BSO predictably inhibited production of glutathione without regard to cysteine source.