FIGURE 5.

NEDD4L regulates the activities of RhoA, Rac1, and JNK. A, NEDD4L knockdown decreases RhoA activity. HEK293T cells were transfected with nonspecific (NS) shRNA or NEDD4L shRNAs. At 40 h post-transfection, the cells were serum-starved for 20 h and then treated with control medium or Wnt5a medium for 4 h. The cells were then lysed, and active GTP-bound RhoA was immunoprecipitated using purified GST-RBD protein and detected by anti-RhoA antibody. Total protein expression was confirmed by immunoblotting with whole cell lysates (WCL). B, NEDD4L knockdown increases Rac1 activity. As in A, except that active GTP-bound Rac1 was immunoprecipitated using purified GST-PBD protein and detected by anti-Rac1 antibody. C, NEDD4L knockdown increases JNK activity. HEK293T cells were transfected, serum starved, and treated with control medium or Wnt5a medium as in A. Then cells were harvested for immunoblotting (IB). Tubulin serves as a loading control. D, NEDD4L knockdown increases AP1 luciferase-reporter activity. HEK293T cells were transfected with NS or NEDD4L shRNA together with AP-1 reporter and Renilla. The cells were then serum-starved and treated with control medium or Wnt5a medium as in A and harvested for luciferase assay. The experiment was performed in triplicate, and data represented as the mean ± S.D. after normalized to Renilla activity (**, p < 0.01).