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Fig. 5. Rgf3p is a specific Rho1-GEF. (A) Rgf3 binds directly to the GDP-bound form of Rho1p (Rho1-T20N). Y190 cells expressing the indicated proteins were cultured on a SD plate with histidine (left) or without histidine plus 40 mM 3AT (right) at 30°C for 3 days. (B) The Rgf3p level modulates the amount of GTP-bound Rho1p in vivo. Wild-type (MS38) cells expressing pREP4X or pREP4X-rgf3, and ehs2-1 (GI 1) mutant cells were transformed with either pREP3XHA-rho1 or pREP3X-HA-rho2. GTP-Rho1p or GTP-Rho2p were pulled down from the cell extracts with GST-C21RBD and blotted against 12CA5, anti-HA monoclonal antibody.





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