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. 2003 Dec;14(12):4958–4970. doi: 10.1091/mbc.E03-06-0426

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Copyright © 2003, The American Society for Cell Biology

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Figure 3. — Rsr1p interacts with Cdc42p. (A) Cdc42p coimmunoprecipitated with HARsr1p from yeast extracts. CDC42, HA-RSR1, and HA-rsr1^T35A were expressed from multicopy plasmids in yeast. Association of Cdc42p and HA-Rsr1p was not detectable when HA-Rsr1p and Cdc42p were expressed at the endogenous level. After immunoprecipitation of HA-Rsr1p (lane 1) or HARsr1^T35Ap (lane 3) with an antibody against the HA epitope, association of Cdc42p was determined by immunoblotting with polyclonal antibodies against Cdc42p (top panel). Approximately equal amounts of HA-Rsr1p and HA-Rsr1^T35Ap were recovered for each reaction as judged by immunoblotting with polyclonal antibodies against Rsr1p (bottom panel). Control reactions without HA antibody were shown in lanes 2 and 4. (B) Association of GST-Rsr1p and His₆-Cdc42p in vitro. GST-Rsr1p (∼400 nM) preloaded with GTPγS (T) or GDP (D) was incubated with His₆-tagged Cdc42p (∼400 nM) preloaded with GTPγS or GDP. Rsr1p-GTPγS + Cdc42p-GTPγS (lane 1); Rsr1p-GTPγS + Cdc42p-GDP (lane 2); Rsr1p-GDP + Cdc42p-GTPγS (lane 3); Rsr1p-GDP + Cdc42p-GDP (lane 4). GST that had been preincubated with GTPγS (lanes 5 and 6) or GDP (lanes 7 and 8) was used as a control. Association of Cdc42p with Rsr1p was determined by immunoblotting with antibodies against Cdc42p (top panel). Approximately equal amounts of GSTRsr1 and GST proteins were recovered for each reaction as judged by immunoblotting with antibodies against GST (bottom panel). (C) Association of Rsr1p and GST-Cdc42p in vitro. Rsr1p (∼400 nM) purified after removal of GST was preloaded with GTPγS (T) or GDP (D) and incubated with GST-Cdc42p (∼400 nM) preloaded with GTPγS or GDP. Rsr1p-GTPγS + Cdc42p-GTPγS (lane 1); Rsr1p-GTPγS + Cdc42p-GDP (lane 2); Rsr1p-GDP + Cdc42p-GTPγS (lane 3); Rsr1p-GDP + Cdc42p-GDP (lane 4). Association of Rsr1p with GST-Cdc42p was determined by immunoblotting with antibodies against Rsr1p (top panel). Approximately equal amounts of GST-Cdc42p were recovered for each reaction as judged by immunoblotting with antibodies against Cdc42p (lower panel). (D) Quantification of the amounts of Rsr1p associated with GST-Cdc42p. Rsr1p preloaded with [³H]GTPγS (T) or [³H]GDP (D) was incubated with GST-Cdc42p preloaded with GTPγS or GDP. The amount of [³H]GTPγS- or [³H]GDP-bound Rsr1p that associated with GST-Cdc42p was determined by a filter binding assay. An average of five independent assays is shown as percentage of counts (cpm) divided by the initial input to each reaction.