CDC42 modulates the interaction between IRSp53 and VASP

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CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP CDC42 modulates the interaction between IRSp53 and VASP. (A) IRSp53 connects VASP with CDC42. Equimolar concentrations (200 nM) of IRSp53and/or VASP were incubated with 1 μM GDP‐ or GTPγS‐loaded GST‐CDC42 immobilized on beads, as indicated. Inputs (10% of total) and bound material were analysed by immunoblotting to visualize VASP or by Ponceau staining to detect IRSp53 and CDC42. (B) Equal amounts of VASP were incubated with Ni‐NTA agarose beads coating with increasing concentrations of recombinant purified His‐IRSp53 (0, 100, 250, and 500 nM), in the absence or presence of saturating concentrations of GTP‐loaded GST‐CDC42. Inputs (10% of total) and bound material were immunoblotted with the indicated antibodies (Ponceau staining visualizes IRSp53). Right panel: Quantification of the fraction of VASP bound to His‐IRSp53‐Ni‐NTA beads was determined by analysing blots with the ImageJ software. The fraction (bound/total) of VASP was plotted against the concentrations of IRSp53 (data are the mean±s.d. of three independent experiments). (C) Lysates (2 mg) of HeLa cells, transfected with empty vector as a control (EV) or with either CDC42 dominant‐negative (N17) or constitutively active (L61) mutants, were immunoprecipitated with an anti‐VASP (VASP) or control antibody (CTR). Input lysates (40 μg) and immunoprecipitates (IPs) were immunoblotted with the indicated antibodies. Right panel: The amount of IRSp53 bound to VASP was determined using the ImageJ software. Relative binding between IRSp53 and VASP (data are the mean±s.d. of five independent experiments).Source data for this figure is available on the online supplementary information page. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 32, Issue: 20, Pages: 2735-2750, First published: 27 September 2013, DOI: (10.1038/emboj.2013.208)