Fig. 3. Expression of IRSp53 and IRTKS in COS7 cells. COS7 cells were transfected with Myc-tagged IRSp53 and IRTKS constructs and 24 hours later fixed and stained with 9E10 and Alexa Fluor 488-anti mouse and TRITC-phalloidin. Left hand panels show staining of the Myc-tagged proteins and the right hand panels show the same field of view with F-actin stained. Cells expressing Myc-IRSp53 exhibit long filopodia-like extensions (A,B). Cells expressing low levels of Myc-IRTKS exhibit short actin microspikes (C,E), which at higher expression levels coalesce into clusters of short spikes (D,F). Confluent cells expressing Myc-IRTKS have a high concentration of clustered actin bundles at cell junctions (G,H). Cells expressing Myc-IRTKS with the Ct extension removed produce long wavy filopodia-like extensions and not actin clusters (I,J), whereas cells expressing Myc-IRSp53 with IRTKS Ct extension fused produce actin clusters (K,L). Boxed insets are magnifications of the indicated region of the image and highlight actin structures typically induced by the construct. Bars, 20 µm. The images shown are representative of more than three independent transfections per construct, where more than 100 cells have been viewed per transfection. For IRSp53, over 80% of cells have the described phenotype of long filopodial protrusions, and for IRSp60, 30-40% of cells show no change in shape, whereas 60-70% have short actin clusters and spikes. Bars, 20 µm.