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Fig. 10. Ras proteins were not properly regulated in sodC– cells. (A) Active Ras proteins are visualized by GFP-RBD signals on the plasma membrane. Wild-type cells often displayed well-organized RBD signal on one side of a cell, whereas sodC exhibited broad GFP-RBD signal around cellular peripheries. Images were captured after 4 hours of cAMP pulsing. (B) A significantly higher basal level of active Ras was detected in extracts from sodC– cells compared with wild-type cells after 4 hours of pulsing. (C) Cells were pulsed with 50nM cAMP for 4 hours, and then stimulated with 10 µM cAMP as indicated. Total Ras protein levels were first normalized by western blot using anti-Pan-Ras antibody. Active Ras proteins were determined by GST-RBD assay (Sasaki et al., 2004). (D) A higher basal level of active Ras in sodC– cells was significantly decreased upon depletion of superoxide by incubation with radical scavenger XTT (4 mM) for 10 minutes at room temperature. As in C, wild-type cells showed a lower level of active Ras. (E) sodC– cells displayed higher basal level of active GFP-RasG than did wild-type cells. (F) GFP-RasG was modestly activated by conditioned medium (CM) and was susceptible to XTT in wild-type cells. In sodC– cells, GFP-RasG showed higher basal activity and was susceptible to XTT, but no further stimulation of GFP-RasG was observed with CM.
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