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. 2004 Jul 1;23(14):2811–2820. doi: 10.1038/sj.emboj.7600287

Figure 6.

Figure 6

14-3-3 inhibits AKAP-Lbc Rho-GEF activity in vitro. (A) HEK-293 cells expressing Flag-AKAP-Lbc were serum starved for 24 h. Lysates were then subjected to immunoprecipitation with anti-Flag monoclonal antibodies. Immunoprecipitates were incubated with purified PKA and then incubated with GDP-loaded RhoA, [35S]GTPγS and concentrations of His-tagged 14-3-3β ranging from 5 to 500 nM. Exchange reactions were terminated after 25 min. (B) Immunoprecipitates of Flag-AKAP-Lbc were incubated with CIP (closed symbols) or with purified PKA (open symbols) and then incubated with GDP-loaded RhoA, [35S]GTPγS and 200 nM of His-tagged purified RIIα (circles) or 14-3-3β (squares). Exchange reactions were terminated after 25 min. (C) Immunoprecipitates of Flag-tagged AKAP-Lbc (circles) or AKAP-Lbc S1565A (squares) were incubated for 1 h with purified PKA and then incubated with GDP-loaded RhoA, [35S]GTPγS and 200 nM of His-tagged RIIα (closed symbols) or 14-3-3β (open symbols). Exchange reactions were terminated after 25 min. Results are representative of three independent experiments.