EVH1 missense mutations show increased actin polymerization in vitro but equivalent Cdc42 binding compared with WASp^WT. (A) Structure of the WASp protein showing main functional domains and site of missense mutants generated for this study. (B) In vitro actin polymerization assay. Purified GST-WASp immobilized on Sepharose beads was incubated in U937 cell lysate supplemented with 5mM MgCl₂ and 1mM ATP. Beads were resolved by SDS-PAGE and Western blotted for GST and actin. A representative experiment with 3 WASp concentrations for each construct is shown. All samples were performed simultaneously as part of a single experiment, but resolved on 2 blots (shown separately). (C) Summary of densitometry of GST-WASp and actin bands from 25 independent experiments. Differences between constructs were analyzed by ANCOVA of actin per unit GST-WASp (compensating for experiment and bead volume) and are expressed as the sympercentage difference in activity from WASp^WT. Error bars represent SEM, and dotted lines show SEM of WT activity. Numbers of experimental samples analyzed per construct are as follows: WASp^WT 49, WASp^T45M 18, WASp^R86H 19, WASp^R138P 22, WASp^A134T 22, WASp^dEVH1 25, WASp^dVCA 19, WASp^S272P 19, WASp^CytD 10. WT Cyt: WASp^WT with 5nM cytochalasin D in lysate. (D) GST-WASp immobilized on Sepharose beads was incubated with U937 cell lysate supplemented with 125mM of the constitutively active Cdc42 12V mutant for 1 hour. Beads were resolved by SDS-PAGE and Western blotted for Cdc42 and GST. A representative blot from 3 experiments is shown. Nonadjacent lanes on Western blots are separated by solid gray lines. All lanes within each figure were on the same gel. ***P < .005, **P < .01, *P < .05, and ns indicates P > .05.