Fig. 8.

Expression of mouse KuzDN blocks Slit-dependent recruitment of Sos to the plasma membrane. HEK293T cells were transiently transfected with human Myc-His-ROBO1. Endogenous human SOS is visualized by rabbit anti-mSos1 antibodies (green), human ROBO1 is visualized by mouse anti-Myc antibodies (blue), and F-actin is visualized by rhodamine-conjugated phalloidin (magenta). A single confocal section is shown. (A-D) A ROBO1-expressing cell treated with control medium. The morphology of the cell is elongated and SOS is predominantly localized in the cytoplasm. (E-H) A ROBO1-expressing cell treated with SLIT2-conditioned medium for five minutes. The cell morphology has become remarkably rounded and SOS proteins are recruited to the membrane, partially colocalizing with ROBO1 (white in H). (I-L) Cells expressing both ROBO1 and mouse KuzDN treated with SLIT2 medium. The cells retain their elongated morphology and endogenous SOS localization is cytosolic. (M) Quantification of the ratio of the average pixel density of SOS staining colocalized with the membrane to the average pixel density of cytosolic SOS. Shown is the average of the ratio of membrane to cytosolic SOS. *P<0.0001 (unpaired Student's t-test) compared with ROBO1-expressing cells treated with bath application of SLIT2. Error bars indicate s.d.