561862.fig.007a
(a)
561862.fig.007b
(b)
561862.fig.007c
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(d)
Figure 7: p-BQ mimics AECS in activating EGFR in a time-dependent manner. Serum-starved A549 cells were nontreated (NT) or exposed to 100 ng/mL EGF for 5 min, 2 μL/mL AECS or 200 ng/mL p-BQ for 15 min, 30 min, or 1 hr, respectively (a). Vitamin C (40 μg/mL) pretreatment of A549 cells for 15 min in serum-free media completely prevented AECS/p-BQ-induced EGFR activation (b). 500 nM Tyrphostin AG1478 (EGFR inhibitor) pretreatment of A549 cells for 15 min in serum-free media completely prevented activation of EGFR by 100 ng/mL EGF, 2 μL/mL AECS, or 200 ng/mL p-BQ (c). Anti-p-BQ antibody prevented AECS/p-BQ-induced EGFR activation (d). Cells were lysed, and the EGFR was immunoprecipitated (IP) from the cell lysates using anti-EGFR antibody. Immunoprecipitated proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted (IB) with either antiphosphotyrosine (PY20) antibody (a, b, c) or with anti-p-BQ antibody (d).