(A) Mutations in the Rac effector domain abolish
plexin-B1 interaction. Various myc-Rac mutants were transfected into
HEK293 cells, and cell lysates were subjected to in
vitro pulldown by MBP-plexin-B1. Expression of different Rac
mutants in cell lysates is shown (lower). The mutations Y32F (lane 5),
T35S (lane7), and F37L (lane 9) in the Rac effector domain completely
abolished interaction with plexin-B1. In contrast, the mutations P34G
(lane 6) and Y40C (lane 9) had no significant effect on the interaction
with plexin-B1. (B) Cdc42 and RhoA do not interact with
plexin-B1. Various mutants of myc-RhoA, -Cdc42, and -Rac were
transfected into HEK293 cells. Cell lysates were used in either
MBP-plexin-B1 or GST-PAK-RBD pulldown. Neither RhoL63 (lane 2) nor
Cdc42L61 (lane 4) bind to plexin-B1. As a control, GST-PAK-RBD binds
both RacL61 (lane 9) and Cdc42L61 (lane 11).