FIGURE 6.

Effect of Akt on GIT1/eNOS/NO signaling. A, sinusoidal endothelial cells were isolated from normal rat livers and transfected with cDNA encoding a dominant active Akt (Akt-CA), a dominant-negative Akt (Akt-DN), or an EV, all 1 μg, and nitrite levels were measured in conditioned medium (n = 3, *, p < 0.005 compared with control; **, p < 0.01 compared with control). B, GIT1 was immunoprecipitated (IP) from normal sinusoidal endothelial cell lysates transfected with Akt-CA or EV followed by immunoblotting (IB) to detect either phospho-eNOS or eNOS (representative immunoblots are shown). C, sinusoidal endothelial cells were transfected with Akt-CA, Akt-DN, or EV as in A, and in the upper panel, lysates were immunoblotted to detect eNOS. In the middle panel, lysates were immunoprecipitated with anti-GIT1, then immunoblotted to detect eNOS; specific eNOS bands were quantitated and normalized to the level of immunoprecipitated GIT1 and presented in the graph in the lower panel (n = 3, *, p < 0.05 versus EV or Akt-DN). D, sinusoidal endothelial cells were transfected with Akt-CA or Akt-DN; representative examples (of >10 others) of immunolocalization of eNOS (left, green) and GIT1 (middle, red) in sinusoidal endothelial cells are shown. Merged images after overexpression Akt-CA, Akt-DN, or EV are shown. The bar is 10 μm in length. E, sinusoidal endothelial cells were transfected with GIT1 siRNA (50 nm) and then with adenovirus (multiplicity of infection of 250) encoding constitutively active Akt (Ad-myrAkt) or empty adenovirus without a cDNA insert (left lane in all immunoblots). After 48 h, cell lysates were subjected to immunoblotting with the indicated antibodies. A scrambled siRNA was used as a negative control siRNA (left and middle lanes in all immunoblots). Specific bands corresponding to phospho-eNOS, GIT1, and phospho-Akt were quantitated and presented graphically (n = 3, *, p < 0.05 versus Ad-myrAkt alone, #, p < 0.001 versus empty virus alone).