Fig. 6. The activation state of the RhoA-GTPase. (A) Immunoblots of control pull-downs using the human form of rhotekin-RBD showing that it is specific for the activated form of RhoA. Approximately 10⁴ SW13 cells were seeded in 5 cm dishes and left untreated or transfected with the vector as indicated above the lanes for 24 hours prior to the pull-down. Myc-tagged mutant forms of RhoA were detected using the anti-myc-9E antibody. (B) The activation states of endogenous RhoA in SW13 cells, a BRG1-expressing clone and a BRG1-K798R-expressing clone were measured 24 hours after seeding at 60% confluence. The protein concentration of each sample was measured in the crude lysates and the protein input adjusted accordingly. The RhoA protein was detected using anti-RhoA antibodies. (C) The activation state of RhoA in SW13 cells, BRG1 and BRG1-K798R-expressing clones seeded at 60% confluence, starved for 18 hours in serum-free medium and stimulated with 5% serum for 20 minutes as indicated above the lanes is shown. (The experiment presented is representative of three experiments.)