FIGURE 7.

Elmo2 modulates Rac1 activation by insulin in L6 cells. A, ectopic expression of Elmo2 in L6 cells enhances Rac1 activity induced by insulin. L6 cells were transduced with adenoviral vectors coding for either GFP or FLAG-Elmo2. Then total cell lysates were prepared after these cells were serum-deprived for 6 h, followed by a 10-min insulin treatment, and subjected to GST pull-down assays with recombinant GST-CRIB. The levels of Rac1 associated with GST-CRIB beads were analyzed by Western blotting with anti-Rac1 antibody (i). iii shows the levels of GST-CRIB in each sample. ii, iv, v, and vi show the level of phospho-Akt, Akt, and Elmo2 in total cell lysates. B, densitometric analyses of Rac1 associated with GST-CRIB beads in A. The amount of Rac1 on GST-CRIB beads from GFP-expressing cells without insulin treatment was set as 1 after normalization to total cellular levels of Rac1. The bar graphs show means ± S.D. (error bars) (n = 3). In all cases, p < 0.034. C, knockdown of Elmo2 in L6 cells reduces Rac1 activity induced by insulin. The experiments were carried out in a manner similar to that described for A except that L6 cells were transduced with adenoviral vectors expressing either luciferase or Elmo2 shRNA. D, densitometric analysis of Rac1 associated with GST-CRIB beads in C. The amount of Rac1 on GST-CRIB beads from luciferase shRNA-expressing cells without insulin treatment was set as 1 after normalization to total cellular levels of Rac1. The bar graphs show means ± S.D. (n = 3). In all cases, p < 0.042.