FIGURE 6.
The interaction between ClipR-59 and Elmo2 is regulated by insulin. A, GST pulldown assay to show that active RhoG and Rac1 enhance ClipR-59 and Elmo2 interaction. The total cell lysates from COS-7 cells that were transiently co-transfected with Myc-Elmo2 expression vectors plus either pEBG (GST vector) or pEBG-ClipR-59 with either constitutive active RhoGG12V or Rac1G12V were subjected to a GST pulldown assay. The proteins associated with GST beads were analyzed in a Western blot with anti-FLAG antibody (i). Panels ii and iii show the levels of Myc-Elmo2 and GFP-tagged RhoG or Rac in TCL. Panel iv shows the levels of GST fusion proteins associated with GST beads. B, insulin and IGF enhances the interaction between Elmo2 and ClipR-59. COS-7 cells were co-transiently transfected with pEBG-ClipR-59 and Myc-Elmo2. 24 h post-transfection, cells were serum-deprived overnight. After treatment with insulin (100 nm) or IGF (5 ng/ml) for 15 min, the cell total lysates were prepared for a GST pulldown assay. The GST beads were analyzed in Western blot with anti-Myc antibody (i). The TCL were analyzed in Western blot with anti-Myc (ii), anti-phospho-Akt (iii), and anti-GST (iv) antibodies, respectively. C, similar to B, except ΔN2 Elmo2 was also used and the cells were only treated with insulin. All of these experiments were repeated three times with similar results. Where necessary, molecular mass markers are shown on the left of the panel. IB, immunoblot.