OL-pc recruits Nap1 and WAVE1 to cell–cell contacts. (A, left) Control (vain vector transfected) U251 cells and those stably expressing OL-pc-HA or OL-pcΔNBS-HA were cultured in a low density and double immunostained for Nap1 and HA tag. Arrows point to Nap1-positive lamellipodia, and arrowheads here as well as in B and C indicate cell–cell contact sites. Note that Nap1 is localized at cell–cell contacts in the transfectants of OL-pc but not in those of OL-pcΔNBS. (right) Double staining for F-actin and HA. Both OL-pc and OL-pcΔNBS colocalize with F-actin in the lamellipodia. (B) Cells in confluent cultures were double immunostained for Nap1 and HA after extraction with 0.5% Triton X-100. (C) WAVE1-GFP was transiently introduced into cells followed by double immunostaining for OL-pc and GFP. Note the accumulation of WAVE1 at OL-pc–positive but not OL-pcΔNBS–positive cell–cell contact sites. Images were taken with a CCD camera for A and B and with a confocal microscope for C. Bars, 20 μm.