FIG. 6. Pervanadate, an agent which induces oxidative stress, stimulates tyrosine phosphorylation of GRP-75; diphenylene iodonium (DPI), an inhibitor of NADH/NADPH oxidase suppresses Ang II-medaited action.

A, Serum starved cells were left untreated (control), or treated with Ang II (100 nM) for 15 min, or interleukin-6 (IL-6; 20 ng/ml) for 15 min, or EGF (100 ng/ml) for 5 min, or pervanadate (3 mM H₂O₂ together with 1 mM vanadate) for 15 min. Cell lysates were prepared and proteins run on an 8% SDS-polyacrylamide gel and immunoblotted with phospho-specific anti-caveolin-1 antibody cross-reactive to the 75 kDa protein (phopshorylated GRP-75). B, the blot in A was stripped and reprobed with anti-Stat3 antibody (loading control). C, Effect of DPI on Ang II-mediated GRP-75 tyrosine phosphorylation. Cells were left untreated, or treated with Ang II alone (100 nM) for 15 min, or DPI alone (10 μM) for 60 min, or first with DPI (10 μM) for 45 min followed by Ang II (100 nM) for 15 min. Cell lysates were prepared and equal amounts of protein were immunoblotted with anti-phospho-caveolin-1 antibody cross reactive to phosphorylated GRP-75. D, the blot in C was stripped and reprobed with anti-stat3 antibody. These blots are representative of four independent experiments.