Fig 9.

AKAP-Lbc and the AKAP-Lbc W2328L mutant display similar RhoA-activating and PKCη binding properties. (A) HEK293 cells were transfected with the HA-tagged α1b-AR in combination with either empty vector or the plasmids encoding the Flag-tagged forms of AKAP-Lbc or AKAP-Lbc W2328L. After a 24-h serum starvation, cells were treated with 10⁻⁴ M PE for 15 min or left untreated. Cell lysates were incubated with GST-RBD beads. The bound RhoA was detected with a monoclonal anti-RhoA antibody (top). The relative amounts of total RhoA, HA-α1b-AR, and Flag-AKAP-Lbc proteins in the cell lysates were assessed by Western blotting as indicated. Results are representative of three independent experiments. (B) HEK293 cells were transfected with the empty Flag vector or the plasmids encoding the Flag-tagged forms of AKAP-Lbc or AKAP-Lbc W2328L. Cell lysates were subjected to immunoprecipitation with anti-Flag antibodies. Western blots of the immunoprecipitates and of the cell extracts were revealed using anti-PKCη polyclonal antibodies to detect endogenous PKCη (top and middle) or anti-Flag monoclonal antibodies to detect the Flag-tagged AKAP-Lbc proteins (bottom). Results are representative of three independent experiments.