Fig. 3. The inactive VASP mutants ΔB and DC are interfering mutants. (A) Interference with VASP-induced SRF activation. NIH 3T3 cells transfected with the SRF reporter expressed intact VASP (0.15 µg) or the indicated VASP mutants (0.1, 0.3 and 0.9 µg). The structure of the mutants is shown below. (B) Interference with VASP-induced F-actin accumulation. Cells expressing intact VASP (0.75 µg) and the indicated VASP mutants (4.5 µg) were analysed for mean cellular F-actin content relative to that of untransfected cells in the same population using FACS. (C) The VASP EVH2 block C is sufficient to bind intact VASP. Lysates from cells expressing the indicated VASP mutants were separated by SDS–PAGE (12.5% gel), transferred to a membrane, and probed either with anti-Flag antibodies (upper panel) or with ³⁵S-labelled VASP DC (residues 278–380), produced by in vitro translation (lower panel).