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We show here that after EGF stimulation, N\u2010WASP is recruited to the nucleation zone of the dynamic leading edge compartment of carcinoma cells, with maximal recruitment of N\u2010WASP within 1 min after EGF stimulation. The timing of N\u2010WASP recruitment mirrors the timing of barbed\u2010end formation at the leading edge. To determine the cellular activation of N\u2010WASP after EGF stimulation, we made a conformation\u2010sensitive antibody (CSA) against the CRIB domain of N\u2010WASP that is predicted to recognize N\u2010WASP in its open, active conformation, but not in its closed, inactive conformation. The ability of CSA to detect only active N\u2010WASP was demonstrated by in vitro experiments using immunoprecipitation of active N\u2010WASP from EGF\u2010stimulated cells and Cdc42 activation of N\u2010WASP activity. In cell staining experiments, N\u2010WASP is maximally accessible to CSA 40 sec after EGF stimulation and this activated N\u2010WASP is in the nucleation zone. These results indicate that active N\u2010WASP is present at the leading edge of lamellipods, an unexpected finding given its reported involvement in filopod formation. This work establishes the feasibility of using antibodies directed against specific conformations or epitopes with changing accessibilities as a window on the status and localization of activity. Cell Motil. Cytoskeleton 59:141\u2013152, 2004. \u00a9 2004 Wiley\u2010Liss, Inc.<\/jats:p>","DOI":"10.1002\/cm.20030","type":"journal-article","created":{"date-parts":[[2004,9,14]],"date-time":"2004-09-14T04:20:25Z","timestamp":1095135625000},"page":"141-152","source":"Crossref","is-referenced-by-count":22,"title":["Cellular localization of activated N\u2010WASP using a conformation\u2010sensitive 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