Figure 7

scFv C1-N1N2 specifically recognizes GTP-bound Rho in vitro and in vivo. A: scFv C1-N1N2 immunoprecipitation (IP) of GTP-bound RhoA and RhoB. Crude bacterial lysates of recombinant RhoA and RhoB loaded with either GTP or GDP were incubated with scFv C1-N1N2 fixed on Ni-beads. An irrevelant scFv was used as control. Complexes on beads were resolved by SDS-PAGE and immunobloted with anti-RhoA and anti-RhoB. Western Blot is representative of 2 independent experiments. B: Immunofluorescence shows that scFv C1-N1N2 specifically binds to activated HeLa cells. Suspension containing scFv C1-N1N2 was incubated with GDP-loaded RhoA(A and B) or GTPγS-preloaded RhoA beads (C). Twenty-four hours after seeding, HeLa cells were serum-starved for 48 h and activated with 10% SVF and EGF (100 ng/ml) for 1 hour. Cells were fixed, permabilized and incubated with supernatants from scFv C1-N1N2-Rho incubation and anti-c-myc FITC conjugate secondary antibody. (A) Non-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads, (B) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads (C) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GTPγS-loaded RhoA beads. (D) Non-activated HeLa cells incubated with the commercial Rhoa antibody, (E) EGF-activated HeLa cells incubated with the commercial RhoA antibody, (F) EGF-activated HeLa cells incubated with irrelevant scFv (anti-tyroglobulin). Pictures are representative of 2 independent experiments.