Active Neural Wiskott-Aldrich syndrome protein (N-WASP) was labeled using a conformation-specific rabbit anti-active N-WASP antibody (1:2000), a gift from John Condeelis and described previously.24 (link) Chicken anti-NeuN (Millipore (Billerica, MA, USA) catalog number ABN91 RRID:AB_11205760) was used to label neuronal somas. Quantification of active N-WASP intensity was accomplished by analyzing a single image in the middle of each individual cell’s Z-plane projection, where the middle was defined as the region in which the soma size is at its maximum. This approach prevents cells with greater spread into the Z-plane from artificially increasing fluorescent intensity measurements as detailed previously.23 (link) Within each experiment, all microscope parameters, including magnification, pinhole size, digital gain and offset, were kept constant. All imaging and quantification was performed blind to experimental conditions.