Fig. 2.

E2 activates cPLA₂ through MMP- and HB-EGF-dependent trans-activation of EGFR. A and B, Western blot analysis of cPLA₂α phosphorylation at residue Ser505 (A) and ERK1/2 MAPK phosphorylation at residues Thr202/Tyr204 (B) was performed on MCF-7 cells treated with either vehicle (0) or 10 nm E2, with or without CRM197 (200 ng/ml, 2 h pretreatment) at the indicated time points. A line separates noncontiguous lanes on the same gel. C, Phosphorylation of cPLA₂α at residue Ser505 was assessed by immunoblotting in MCF-7 cells treated with either vehicle (control) or 10 nm E2 for 1 min, with or without the MMP inhibitor GM6001 (10 μm). D, Phosphorylation of cPLA₂α at residue Ser505 was assessed by immunoblotting in MCF-7 cells treated with either vehicle (0) or 10 nm E2 for 1 and 10 min, with or without the c-Src antagonist PP2 (100 nm). E, Phosphorylation of ERK1/2 at residues Thr202/Tyr204 was assessed by immunoblotting in MCF-7 cells treated with either vehicle (0) or 10 nm E2 for 10 min, with or without the c-Src antagonist PP2 (100 nm). All blots were stripped and reprobed with total cPLA₂ or total ERK1/2 MAPK antibodies. Total cPLA₂α or total ERK1/2 was used for protein level normalization as appropriate. Densitometric analysis of three independent experiments is shown with a representative blot. Data are mean values ± se. *, P < 0.01 compared with E2 stimulation.