FIGURE 6.

Effect of pervanadate on luciferase activity in the wild-type EGF receptor. A, CHO cells stably expressing wild-type EGF receptors were preincubated with 200 μm sodium pervanadate for 20 min and then 10 nm EGF was added for the times indicated. Cells were immediately lysed in RIPA buffer, and equal amounts of protein lysates were separated on an SDS-PAGE gel. Western blotting was performed with the indicated antibodies. B, anti-phosphotyrosine Western blots from A were quantitated using Image J, and the level of EGF receptor autophosphorylation was normalized to the maximum phosphorylation observed in cells expressing wild-type receptors. C and D, cells stably expressing EGFR-NLuc and EGFR-CLuc were plated in 96-well plates 48 h prior to assay. On the day of imaging, cells were pretreated for 20 min with 0.6 mg/ml d-luciferin in the absence or presence of 200 μm sodium pervanadate. 10 nm EGF was then added to the cells, and the change in photon flux was monitored over time in untreated cells (C) or pervanadate-treated cells (D).