FIGURE 3.

Biochemical features of the GIT1 and eNOS interaction. A, immunoprecipitation (IP) of recombinant GIT1 or eNOS (r-GIT1, 2 μg; r-eNOS, 1 μg) reveals that eNOS binds GIT1 (upper panels) or GIT1 binds eNOS (lower panels). Immunoprecipitation with IgG as a control is also shown (upper left and lower left panels). B, the activity of r-eNOS (2 μg) was examined after incubation without or with r-GIT1 (4 μg) in the presence or absence of l-NAME (1 mm). The activity of r-eNOS alone was arbitrarily set to 100; n = 3, *, p < 0.005, and **, p < 0.001 compared with r-eNOS alone. C, r-eNOS (2 μg) was incubated with vehicle (control) or r-GIT1 (2 μg) in the presence of 10 μm Ca²⁺ and 100 nm CaM, and eNOS activity was determined as a function of l-arginine concentration (n = 3, *, p < 0.001 and **, p < 0.01 compared with r-eNOS alone). D, r-eNOS (2 μg) was incubated with vehicle (control) or r-GIT1 (2 μg) in the presence or absence of CaM (100 nm), and then NADPH-dependent cytochrome c reductase activity was determined at 23 °C for 3 min (n = 3, *, p < 0.01, and **, p < 0.1 compared with r-eNOS alone). E, GST fusion proteins containing fragments of individual GIT1 domains (4 μg) were incubated with r-eNOS (2 μg) and eNOS activity was measured (note, GST-GIT1 encoding full-length GIT1 is misfolded and insoluble when expressed in E. coli and so was not tested). The activity of eNOS + GST alone was arbitrarily set to 100; n = 3, *, p < 0.05 and **, p < 0.01 compared with eNOS + GST alone. F, schematic diagram showing the GIT1 domains and highlighting specific GIT1 mutations. G, the indicated FLAG-tagged constructs were transfected into normal sinusoidal cells and NOS activity was measured in cell lysates after normalization to control (EV) (n = 3, *, p < 0.01 versus EV). IB, immunoblot.