Fig. 2. LPR1 localizes to and increases the number of actin-rich membrane protrusions in HeLa and Cos7 cells. Cos7 cells were transfected with pEGFP, pEGFP-LPP3 (A) and pEGFP-LPR1 (B) and the distribution of EGFP-tagged protein and actin organization were visualized by immunofluorescence microscopy. (C) Bar graph comparing the number of filopodia, dorsal and peripheral, as visualized by phalloidin staining, in untransfected (control) Cos7 cells and Cos7 cells expressing either EGFP or EGFP-LPR1. Twenty cells were counted for each category; error bars denote s.d. for each group. (D). HeLa cells were transfected with pEGFP-LPR1 and localization of the protein and actin organization were determined by fluorescence microscopy. All images were acquired and processed identically. Panels B and D contain higher magnification images of sections of the cell periphery in the images above to show actin staining and LPR1 localization to filamentous structures. (E) Cell lysates from HeLa cells transfected with pEGFP, pEGFP-LPP3 or pEGFP-LPR1 were examined by western blotting with a GFP-specific antibody. Each lane was loaded with equal amounts of total protein. (F) Cos7 cells were transfected with pEGFP-LPR1, fixed, counterstained with Rhodamine phalloidin, and analyzed by confocal microscopy. Images are shown as projections of z sections in both Rhodamine and GFP channels. (G) Cos7 cells were transfected with pEGFP-LPR1, and examined live by confocal microscopy. The image represents a projection of z sections at a single time interval in a GFP-specific channel. Bars, 10 µm.