Journal: Molecular Endocrinology

Article Title: Estrogen-Induced Activation of Erk-1 and Erk-2 Requires the G Protein-Coupled Receptor Homolog, GPR30, and Occurs via Trans-Activation of the Epidermal Growth Factor Receptor through Release of HB-EGF

doi: 10.1210/me.14.10.1649

Figure Lengend Snippet: Fig. 1. Activation of Erk-1/-2 by Estrogens or Antiestrogens Does Not Correlate with ER Expression Human MCF-7, SKBR3, or MDA-MB-231 breast carcinoma cells, untreated or exposed to 1 nM 17b-estradiol, 1 mM ICI 182,780, or 1 ng/ml EGF for the lengths of time indicated (minutes), were lysed in ice-cold RIPA detergent. Cellular proteins (50 mg) were resolved on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with antibodies specific for phosphorylated Erk-1 and -2. The nitrocellulose membrane was then stripped and reprobed with antibodies that recognize total (phosphorylation state-independent) Erk-2 protein. The relative positions of phosphorylated Erk-1 and -2 and total Erk-2 proteins are designated at the right. The ER expression profile of each cell line is indicated. The data shown above are representative of at least three independent experiments. Below, Band intensities from these individual experiments were quantified using NIH Image software. Results were normalized to total Erk-2 expression in each sample and are plotted with SEM. *, Erk-1/-2 activation significantly (P , 0.05, by Student’s t test) greater than that in unstimulated cells.

Article Snippet: Human MCF-7 (ERa1, ERb1), SKBR3 (ERa2, ERb2), and MDA-MB-231 (ERa2, ERb1) breast carcinoma cells were obtained from American Type Culture Collection (Manassas, Estrogen Action via a G Protein-Coupled Receptor Homolog 1657 D ow nloaded from https://academ ic.oup.com /m end/article-abstract/14/10/1649/2751076 by guest on 26 O ctober 2019 VA) and were cultured in phenol red-free DMEM/Ham’s F-12 medium (1:1) containing 10% FBS and 100 mg/ml of gentamicin.

Techniques: Activation Assay, Expressing, Membrane, Phospho-proteomics, Software

Journal: Molecular Endocrinology

Article Title: Estrogen-Induced Activation of Erk-1 and Erk-2 Requires the G Protein-Coupled Receptor Homolog, GPR30, and Occurs via Trans-Activation of the Epidermal Growth Factor Receptor through Release of HB-EGF

doi: 10.1210/me.14.10.1649

Figure Lengend Snippet: Fig. 2. Estrogen-Induced Activation of Erk-1/2 Requires Cellular Expression of the G Protein Receptor Homolog, GPR30 A, Expression of ERa, ERb, and GPR30 in MCF-7, SKBR3, or MDA-MB-231 cells stably expressing GPR30 or transfected with control vector was assessed by immunoblotting with antibodies specific for ERa, ERb, or GPR30. B and C, Using phosphorylation state-dependent or -independent antibodies, phospho-Erk or Erk expression was determined from whole cell lysates of MDA-MB-231 (GPR30) cells that were untreated, stimulated with EGF (1 ng/ml; 10 min), or exposed to 17b-estradiol (1 nM) or ICI 182,780 (1 mM), or to 17a-estradiol (1 nM), 4-hydroxytamoxifen (1 mM), or progesterone (1 nM) for the indicated times (minutes). The positions of phosphorylated Erk-1/-2 and total Erk- 2 proteins are indicated at the right. The data shown are representative of at least three independent experiments. As in Fig. 1, band intensities from independent experiments have been quantified by densitometry and are plotted with the SEMs. *, Erk-1/-2 activation significantly (P , 0.05, by Student’s t test) greater than unstimulated cells.

Article Snippet: Human MCF-7 (ERa1, ERb1), SKBR3 (ERa2, ERb2), and MDA-MB-231 (ERa2, ERb1) breast carcinoma cells were obtained from American Type Culture Collection (Manassas, Estrogen Action via a G Protein-Coupled Receptor Homolog 1657 D ow nloaded from https://academ ic.oup.com /m end/article-abstract/14/10/1649/2751076 by guest on 26 O ctober 2019 VA) and were cultured in phenol red-free DMEM/Ham’s F-12 medium (1:1) containing 10% FBS and 100 mg/ml of gentamicin.

Techniques: Activation Assay, Expressing, Stable Transfection, Transfection, Control, Plasmid Preparation, Western Blot, Phospho-proteomics

Journal: Molecular Endocrinology

Article Title: Estrogen-Induced Activation of Erk-1 and Erk-2 Requires the G Protein-Coupled Receptor Homolog, GPR30, and Occurs via Trans-Activation of the Epidermal Growth Factor Receptor through Release of HB-EGF

doi: 10.1210/me.14.10.1649

Figure Lengend Snippet: Fig. 4. EGF Receptor Kinase Activity Is Required for GPR30-Mediated Erk-1/-2 Activation A, MDA-MB-231 (GPR30) cells were treated in the absence or presence (15 min) of tyrphostins AG-1478 or AG-879 (50 mM) or the Src kinase inhibitor, PP2 (50 mM), before stimulation for 5 min with 17b-estradiol (1 nM), ICI 182,780 (1 mM), or EGF (10 ng/ml). Phospho-Erk and Erk-2 expressions in these samples were determined as previously described. After immunoprecipitation with the ErbB1-specific antibody, Ab-1, tyrosine-phosphorylated EGFR was detected by immunoblotting with the phosphotyrosine-specific monoclonal antibody, PY20. EGFR recovery was assessed by stripping these membranes and reprobing with ErbB1-specific antibodies. B, MDA-MB-231 or SKBR3 cells were treated with 17b-estradiol (1 nM), ICI 182,780 (1 mM), or EGF (10 ng/ml) for the indicated times (minutes) and lysed in ice-cold RIPA buffer. EGFR tyrosine phosphorylation and recovery were measured as described in A.

Article Snippet: Human MCF-7 (ERa1, ERb1), SKBR3 (ERa2, ERb2), and MDA-MB-231 (ERa2, ERb1) breast carcinoma cells were obtained from American Type Culture Collection (Manassas, Estrogen Action via a G Protein-Coupled Receptor Homolog 1657 D ow nloaded from https://academ ic.oup.com /m end/article-abstract/14/10/1649/2751076 by guest on 26 O ctober 2019 VA) and were cultured in phenol red-free DMEM/Ham’s F-12 medium (1:1) containing 10% FBS and 100 mg/ml of gentamicin.

Techniques: Activity Assay, Activation Assay, Immunoprecipitation, Western Blot, Stripping Membranes, Phospho-proteomics