In Vitro Actin Polymerization Assay.
Actin polymerization was evaluated by assaying increase in fluorescence of pyrene-labeled actin using the actin polymerization kit from Cytoskeleton, Inc. For these assays, cdc42-V12, PTP-PEST, and PSTPIP1 were purified as GST fusion proteins and these proteins or Fyn (Upstate Biotechnology) were added alone or in combination with 1.5× actin polymerization kit buffer containing 20 nM Arp2/3 complex, 100 nM GST-WASp or WASpΔGBD fusion protein, and 100 μl monomer pyrene actin stock in G buffer (5mM Tris-HCl, pH 8.0, 0.2 mM CaCl2, 0.5 mM DTT, and 0.2 mM ATP), with the final concentration of G actin being 2.8 μM. Fluorescence changes were monitored every minute for over 1 h at room temperature using a fluorometer (Photon Technology International) with filters for excitation at 365 nm and emission at 407 nm.