Fig. 6. VASP functions in the Rho–mDia–actin pathway to SRF activation. (A) Functional Rho is required for efficient VASP-induced SRF activation. Cells transfected with SRF reporter expressed intact VASP (0.02, 0.05, 0.15 µg) or VASP mutants (ΔPP: 0.02, 0.05, 0.15 µg; EVH2: 0.05, 0.2, 0.6 µg), together with C3 transferase (0.1 µg) as indicated. (Inset) Immunoblot analysis of VASP protein levels. (B) Functional Rho and mDia1 are required for VASP-induced SRF activation and F-actin accumulation. Left: SRF activation. Cells transfected with SRF reporter and expressing C3 transferase (0.1 µg) or interfering mDia1 mutant F1F2Δ1 (Copeland and Treisman, 2002) (0.9 µg) were processed as in (A). Inset: VASP immunoblot of cell lysates. Right: F-actin accumulation. Cells expressed intact VASP (0.75 µg) with either C3 transferase (0.5 µg) or mDia1 mutant F1F2Δ1 (4.5 µg). Mean cellular F-actin content of the transfected cells was determined relative to that of untransfected cells in the same population using FACS. (C) Functional VASP is required for efficient mDia1- induced SRF activation. Left: cells transfected with SRF reporter expressed activated mDia1 mutants FH1FH2 (0.03 µg) or FH2 (0.3 µg) (Copeland and Treisman, 2002), and VASPΔB (0.1, 0.3 and 0.9 µg). Right: mDia derivatives. Ellipse, Rho-binding domain; D, DAD domain. Immunoblot for mDia1 indicates that VASPΔB expression does not affect mDia1 protein levels.