Fig. 1.

Abi1 is found in invadopodia in MDA-MB-231 cells. (A) Expression and complex formation of Abi1 with Sra, Nap1, WAVE2 and N-WASP. The MDA-MB-231 cells were lysed and immunoprecipitated (IP) with pre-immune serum (Pre-IP) or anti-Abi1-specific antibodies (Abi1-IP), as indicated. The immunoprecipitates, along with total cell lysate (TCL), were separated on 8% SDS–polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membranes and subjected to western blot (WB) analysis using the indicated antibodies. (B) Abi1 colocalizes with invadopodia. The MDA-MB-231 cells were incubated with anti-Abi1 specific antibody and subsequently stained with Alexa-conjugated secondary antibodies (green). The cells were then counterstained with Alexa-conjugated phalloidin to visualize F-actin (red). Abi1 location in invadopodia is shown by an arrow in the merged image (merge); bar: 10 μm. (C) Abi1 is found in ECM degradation sites. MDA-MB-231 cells were grown on coverslips coated with a thin layer of FITC-conjugated gelatin (green). Cells were incubated with Abi1 antibody and subsequently stained with Alexa-conjugated secondary antibody (red). Degradation is indicated as dark patches within the fluorescent monolayer (upper panel). Abi1 is found in degraded area, as shown by arrows. (D) Expression of GFP and GFP–Abi1 in MDA-MB-231 cells. The MDA-MB-231 cells were transfected with plasmids expressing GFP alone or GFP-tagged Abi1, as indicated. Total cell lysate containing 50 μg protein was separated on SDS–PAGE and analyzed by western blot using indicated antibodies. (E) GFP–Abi1 is found in invadopodia-like structures. The MDA-MB-231 cells expressing GFP–Abi1 were counterstained with Alexa-conjugated phalloidin (red). The subcellular localization of GFP–Abi1 and F-actin was examined by fluorescence microscopy. GFP–Abi1 was found in F-actin-enriched puncta, as indicated by arrow. (F) F-actin-enriched puncta colocalize with ECM degradation sites. MDA-MB-231 cells were grown on coverslips coated with a thin layer of FITC-conjugated gelatin (green). Cells were stained with Alexa-conjugated phalloidin (red) and examined by fluorescence microscopy. Degradation is indicated as dark patches within the fluorescent monolayer. The F-actin-enriched puncta locate in the degraded area, as indicated by arrows; bar: 10 μm. (G) GFP–Abi1 colocalizes with cortactin. The MDA-MB-231 cells expressing GFP–Abi1 were incubated with anti-cortactin-specific antibody and subsequently stained with Alexa-conjugated secondary antibodies (red). The subcellular localization of GFP–Abi1 and cortactin was examined by fluorescence microscopy. The colocalization of Abi1 with cortactin is shown by the merged image (merge). The arrows indicate Abi1, invadopodia and their colocalization; bar: 10 μm.