Figure 1

Activation of EGFR in human breast cancer cells after nicotine treatment. (A) Human MCF10A and MDA-MB-231 cells were treated with nicotine (0.5 μM) for various times and cell lysates (without nucleus) were extracted for testing EGFR expression by immunoblot. The blot was re-probed with an anti-β-actin Ab for loading control. (B) Expression of EFGR in nicotine-treated MCF10A cells in the presence of MCA (50 nM) was analyzed by immunoblot. The blot was re-probed with an anti-β-actin antibody for loading control. (C and D) MCF10A cells were treated with MCA (50 nM) or AG1478 (100 nM) for 15 minutes prior to 1 hour of nicotine exposure. The phospohrylation pattern of EGFR in nicotine-treated MCF10A cells was examined by immunoprecipitation with anti-EGFR antibody and immunoblotting with the anti-phosphor-tyrosine antibody, or immunoblotting with the anti-phosphor (Tyr 1068) antibody. The blot was re-probed with an anti-EGFR antibody for loading control.(E) With or without treatment with nicotine (left panels) or EGF (right panels), the cell lysates were extracted and precipitated with glutathione S-transferase/growth factor receptor-bound protein 2 (GST/Grb2) fusion protein. Subsequently, the precipitates were subjected to immunoblotting. The blot was also re-probed with anti-Grb2 antibody for the control.