OL-pc associates with the Nap1–WAVE complex. (A) A lysate of newborn mouse brain was incubated with GST or the GST-fused cytoplasmic domain OL-pc (GST–OL-pc). Proteins pulled down with these recombinants were separated by SDS-PAGE and detected by silver staining. Bands of 140 kD and 125 kD were identified as CYFIP2 and Nap1, respectively. (B) Coprecipitation of OL-pc with Nap1 but not with CYFIP2. COS7 cells were transiently transfected with plasmids for FLAG-tagged OL-pc, HA-tagged Nap1, or both (top) or with Myc-tagged CYFIP2 or it and FLAG-tagged OL-pc (bottom), and their lysates were processed for immunoprecipitation (IP) with antibodies against the tags. Vain vectors were used for negative controls (−). Precipitated products separated by SDS-PAGE were detected by Western blotting (WB) with the indicated antibodies. (C) GST fusion proteins containing the OL-pc cytoplasmic domain with various deletions (top) and their interaction with components of the Nap1–WAVE complex (bottom). EC, extracellular; TM, transmembrane; CP, cytoplasmic. CM1 and CM2 represent the portions conserved in this protein family. In the bottom panel, these fusion proteins were incubated with a 1-d-old mouse brain lysate, and the proteins pulled down were analyzed. In the top blot, CYFIP1 (top) and Nap1 (bottom) were detected simultaneously by mixing the antibodies against them. We could not test for CYFIP2 in this assay because of the unavailability of antibodies against this molecule. (D) Schematic diagrams for HA-tagged OL-pc without and with the Nap1-binding region (top) and their interaction with Nap1 (bottom). U251 cells were stably transfected with OL-pc-HA, OL-pcΔNBS-HA, or control vectors, and immunoprecipitates obtained with anti-HA tag antibodies from their lysates were analyzed by Western blotting. α-Tubulin was used for a loading control.