Fig. 1.

Neurite purification assay, biochemical, and proteomic analyses. (A) Schematic of microporous filter system. (B) Neurite lysate protein amount on the filter bottom was determined for the indicated times from filters coated with laminin on the top, the bottom, or both sides. Standard deviations from three independent experiments are shown. (C) Fluorescence micrographs of α-tubulin (red) and F-actin (green, phalloidin) immunostained neurites extending to the lower filter surface for the indicated times. (Scale bar, 20 μm.) (D) 3D reconstruction and volume rendering of a confocal series of α-tubulin stained neurons on filter. (E) Equal amounts of neurite and soma lysates were separated by SDS/PAGE and either silver stained or Western blotted for phosphotyrosine (pY). (F) Rac and Cdc42 activity (GTP-Cdc42, GTP-Rac) was determined from equal amounts of neurite and soma protein fractions, using a GST-PBD pulldown assay and Western blot analysis. ERK served as a protein loading control. (G) Erk activity in neurite and soma fractions was determined by Western blot analysis with phosphospecific antibodies to the phosphorylated activated form of ERK. (H) Gene ontology analysis of the most significant canonical pathways present in the neurite (blue, 10 of 39 shown), soma (yellow), or equally distributed proteins (red). Green dotted line represents significance threshold as measured by Fishers's test (P < 0.05).