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. 2004 Nov 29;101(49):17126–17131. doi: 10.1073/pnas.0407492101

Fig. 4.

Fig. 4.

Disruption of striatin-ERα binding prevents E2-induced nongenomic, but not genomic, signaling. (A) Overexpression of a peptide consisting of the striatin-binding domain within ERα, ER176–253, prevents complex formation between ERα and striatin. (B) Overexpression of the blocking peptide ER176–253 does not interfere with transcriptional transactivation of ERα by E2. Cos1 cells were transiently transfected with an ERα expression plasmid and an estrogen response element-driven luciferase reporter plasmid, with a plasmid encoding ER176–253 or an empty vector. Cells were treated with vehicle alone (open bars) or 10–8 M E2 (filled bars). E2-induced activation of ERα was somewhat greater in the presence of ER176–253. Bars represent mean ± SE from four independent experiments. *, P < 0.05 vs. empty vector. (C) Overexpression of ER176–253 blocks E2-induced increases in phosphorylation of MAPK. Lysates of Cos1 cells transfected with ERα and a control vector, or ERα and ER176–253, were immunoblotted for phospho-MAPK (pMAPK) or total MAPK, after treatment with E2 for various durations. Bar graphs show the mean ± SE for four independent experiments. *, P < 0.01; **, P < 0.05 vs. time 0. (D) Overexpression of the blocking peptide ER176–271 prevents E2-induced phosphorylation of Akt kinase. EAhy926 cells expressing endogenous ERα were incubated with Tat-GFP (○) or Tat-ER176–253 (▪) for 6 h before E2 treatment. Total protein lysates were then immunoblotted for phospho-AKT (pAKT) normalized for the amount of total Akt. These ratios were normalized to one for the vehicle-treated cells. Results represent mean ± SE derived from four independent experiments. *, P < 0.05 vs. time 0. (E) Overexpression of the blocking peptide ER176–253 prevents E2-induced phosphorylation of eNOS. EAhy926 were incubated with Tat-GFP (○) or Tat-ER176–253 (▪) for 6 h before E2 treatment. Total protein lysates were then immunoblotted for phospho-eNOS (peNOS) normalized for the amount of total eNOS. These ratios were normalized to one for the vehicle-treated cells. Results represent mean ± SE derived from four independent experiments. *, P < 0.05 vs. time 0.