FIG. 6.

Pretreatment of cells with EGF inhibits a subsequent arsenite response. (A) PC12 cells were pretreated with EGF (1 μg/ml) for 1 h (+) or not pretreated (−), as indicated at the bottom. The cells were then washed and incubated in EGF-free medium for the time intervals shown at the bottom. The cells were further treated with EGF (100 ng/ml) for 10 min or NGF (100 ng/ml) for 5 min (second treatment, shown at the top). Cell lysates following these different treatments were subjected to Western blot analysis using anti-EGFR polyclonal antibody (upper panel). The same blot was sequentially probed with anti-phospho-ERK-specific antibody (lower panel). (B) PC12 cells were pretreated with EGF (1 μg/ml) for 1 h (+) or not pretreated (−), as indicated at the bottom. The cells were then washed and incubated in EGF-free medium for the time intervals shown at the bottom. The cells were further treated (second treatment) with 400 μM arsenite for an additional 30 min. Total cellular proteins were subjected to Western blot (WB) analysis using anti-phospho-ERK-specific antibody (upper panel) or the antiphosphotyrosine (anti-PY) monoclonal antibody 4G10 (lower panel). EGFR-mediated protein tyrosine phosphorylation stimulated by arsenite is indicated by arrows.