erα gfp expression cassette  (Addgene inc)

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: (A) Immunoblots for ER, PR, and HER2 in different human breast cancer cell lines. ER + (MCF-7 and T-47D) and ER - (MDA-MB-231) breast cancer cell lines were prepared for western blotting. The expression pattern of different hormone receptors was determined. Protein expression levels were compared to expression levels of α-tubulin; each value under the blots indicates relative protein expression levels determined by densitometric analysis, unless specifically mentioned (mean (SD); n = 4). Dots denote ER + (MCF-7 and T-47D) or ER - (MDA-MB-231) breast cancer cells treated with different concentrations of (B) TAM and (C) β-estradiol (E 2 ). Bars denote (D) ER + MCF-7, (E) ER + T-47D, and (F) ER - MDA-MB-231 breast cancer cells treated with TAM after silencing with NC or CNSK1G2 siRNA. (G) ER - MDA-MB-231 also transiently transfected with GFP-C1 or GFP-ERα plasmid DNA as well as with NC or CSNK1G1 siRNA. Twenty-four hours after transfection, cells were treated with vehicle or 1 μM TAM for 24 h. Cellular toxicity was then measured using the MTT assay (mean (SD); n = 4; * P < 0.05, ** P < 0.01, # P < 0.001 vs . each represented counterpart).

Article Snippet: pGFP-C1-ERα (GFP-ERα), a mammalian expression of ERα fused to GFP, was purchased from Addgene (#28230; Watertown, MA, USA).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, MTT Assay

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: Immunoblots for PI3K/AKT/mTOR/S6K and ERK signaling-associated proteins in (A-D) MCF-7, ( E-H) MDA-MB-231, and (I-K) GFP-ERα-overexpressed MDA-MB-231 cells. Western blotting analysis from the breast cells transfected with control siRNA ( NC ) or CSNK1G2 siRNA ( CSNK1G2 ) were performed after treatment with vehicle or 1 μM TAM for 24 h. Protein expression levels based on individual bands of phosphor were compared to expression levels of α-tubulin and/or pan (total) protein. Each value under the blots indicates relative protein expression levels determined by densitometric analysis. Data are presented as mean (SD); n = 4.

Article Snippet: pGFP-C1-ERα (GFP-ERα), a mammalian expression of ERα fused to GFP, was purchased from Addgene (#28230; Watertown, MA, USA).

Techniques: Western Blot, Transfection, Control, Expressing

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: (A) Cytotoxicity assay of either GFP-C1 or GFP-ERα-transfected ER - breast cancer cells. Bar denote relative percentage of % survival of transfected MDA-MB-231 cells after treatment with vehicle, 1 μM TAM, 2 μM D4476, and 1 μM TAM plus 2 μM D4476 for 24h. Cellular toxicity was measured using the MTT assay (mean (SD), n = 5; * P < 0.05, ** P < 0.01 vs . each represented counterpart). (B) Western blotting analysis from GFP-ERα-transfected ER - breast cells treated with vehicle or 2 μM D4476 together with 1 μM TAM for 24 h were performed. Immunoblots for PI3K/AKT/mTOR/S6K signaling-associated proteins in ER - MDA-MB-231 cells. Protein expression levels based on individual bands of phosphor were compared to expression levels of α-tubulin and/or pan (total) protein. Each value under the blots indicates relative protein expression levels determined by densitometric analysis (mean ± SEM, n = 4).

Article Snippet: pGFP-C1-ERα (GFP-ERα), a mammalian expression of ERα fused to GFP, was purchased from Addgene (#28230; Watertown, MA, USA).

Techniques: Cytotoxicity Assay, Transfection, MTT Assay, Western Blot, Expressing

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: (A) Immunoblots for ER, PR, and HER2 in different human breast cancer cell lines. ER + (MCF-7 and T-47D) and ER - (MDA-MB-231) breast cancer cell lines were prepared for western blotting. The expression pattern of different hormone receptors was determined. Protein expression levels were compared to expression levels of α-tubulin; each value under the blots indicates relative protein expression levels determined by densitometric analysis, unless specifically mentioned (mean (SD); n = 4). Dots denote ER + (MCF-7 and T-47D) or ER - (MDA-MB-231) breast cancer cells treated with different concentrations of (B) TAM and (C) β-estradiol (E 2 ). Bars denote (D) ER + MCF-7, (E) ER + T-47D, and (F) ER - MDA-MB-231 breast cancer cells treated with TAM after silencing with NC or CNSK1G2 siRNA. (G) ER - MDA-MB-231 also transiently transfected with GFP-C1 or GFP-ERα plasmid DNA as well as with NC or CSNK1G1 siRNA. Twenty-four hours after transfection, cells were treated with vehicle or 1 μM TAM for 24 h. Cellular toxicity was then measured using the MTT assay (mean (SD); n = 4; * P < 0.05, ** P < 0.01, # P < 0.001 vs . each represented counterpart).

Article Snippet: pGFP-C1-ERα (GFP-ERα), a mammalian expression of ERα fused to GFP, was purchased from Addgene (#28230; Watertown, MA, USA).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, MTT Assay

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: Immunoblots for PI3K/AKT/mTOR/S6K and ERK signaling-associated proteins in (A-D) MCF-7, ( E-H) MDA-MB-231, and (I-K) GFP-ERα-overexpressed MDA-MB-231 cells. Western blotting analysis from the breast cells transfected with control siRNA ( NC ) or CSNK1G2 siRNA ( CSNK1G2 ) were performed after treatment with vehicle or 1 μM TAM for 24 h. Protein expression levels based on individual bands of phosphor were compared to expression levels of α-tubulin and/or pan (total) protein. Each value under the blots indicates relative protein expression levels determined by densitometric analysis. Data are presented as mean (SD); n = 4.

Article Snippet: pGFP-C1-ERα (GFP-ERα), a mammalian expression of ERα fused to GFP, was purchased from Addgene (#28230; Watertown, MA, USA).

Techniques: Western Blot, Transfection, Control, Expressing

Journal: PLoS ONE

Article Title: CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells

doi: 10.1371/journal.pone.0246264

Figure Lengend Snippet: (A) Cytotoxicity assay of either GFP-C1 or GFP-ERα-transfected ER - breast cancer cells. Bar denote relative percentage of % survival of transfected MDA-MB-231 cells after treatment with vehicle, 1 μM TAM, 2 μM D4476, and 1 μM TAM plus 2 μM D4476 for 24h. Cellular toxicity was measured using the MTT assay (mean (SD), n = 5; * P < 0.05, ** P < 0.01 vs . each represented counterpart). (B) Western blotting analysis from GFP-ERα-transfected ER - breast cells treated with vehicle or 2 μM D4476 together with 1 μM TAM for 24 h were performed. Immunoblots for PI3K/AKT/mTOR/S6K signaling-associated proteins in ER - MDA-MB-231 cells. Protein expression levels based on individual bands of phosphor were compared to expression levels of α-tubulin and/or pan (total) protein. Each value under the blots indicates relative protein expression levels determined by densitometric analysis (mean ± SEM, n = 4).

Article Snippet: pGFP-C1-ERα (GFP-ERα), a mammalian expression of ERα fused to GFP, was purchased from Addgene (#28230; Watertown, MA, USA).

Techniques: Cytotoxicity Assay, Transfection, MTT Assay, Western Blot, Expressing

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Estrogen receptors differentially regulate intracellular calcium handling in human nonasthmatic and asthmatic airway smooth muscle cells

doi: 10.1152/ajplung.00206.2019

Figure Lengend Snippet: Effect of specific estrogen receptor (ER) small interference RNA (siRNA) and overexpression on intracellular Ca2+ ([Ca2+]i) response in nonasthmatic human airway smooth muscle (ASM) cells. A: effects of E2, PPT, and WAY in ER-specific siRNA-transfected cells on the AUC of the [Ca2+]i response to histamine were evaluated in nonasthmatic human ASM cells. B: transfection efficiency of siRNA was evaluated using mRNA expression studies. C: ERα and ERβ overexpressed ASM cells were treated with nonselective agonist E2 and their AUC of the [Ca2+]i response to histamine were evaluated. D: representative traces showing effect of overexpression of specific ERs on the [Ca2+]i. E: representative images of ERα-GFP and ERβ-GFP transfections on nonasthmatic ASM cells. **P < 0.01, ***P < 0.001 vs. negative siRNA or lipofectamine (Lipofect). Data are presented as means ± SE; n = 5–7 patients.

Article Snippet: For overexpression studies, ERα and ERβ plasmids tagged with GFP were created and gifted by Dr. Ratna Valdamudi (obtained via Addgene, cat. no. 65211 and 65212).

Techniques: Over Expression, Transfection, Expressing