DMSO, E2 and TAM were obtained from Sigma-Aldrich (St. Louis, MO USA). RAL (Evista®, Eli Lilly and Company, Indianapolis, IN USA) was purchased from the University of Illinois at Chicago Hospital Pharmacy. Cell culture reagents were obtained from Life Technologies (Carlsbad, CA USA). Tissue culture plasticware was purchased from Becton-Dickinson (Franklin Lakes, NJ USA). The following antibodies were used: rabbit polyclonal PKCα (C-20, Santa Cruz Biotechnology, Dallas, TX USA), rabbit polyclonal ERα (SP1, Lab Vision, Thermo Scientific, Kalamazoo, MI USA), mouse monoclonal β-actin (Sigma, St. Louis, MO USA), anti-rabbit Alexa Fluor 488 (Life Technologies, Carlsbad, CA USA) and anti-mouse Cy3 (Jackson Immunoresearch Laboratories, West Grove, PA).
Erα sp1
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
ERα (SP1) is a lab instrument produced by Thermo Fisher Scientific. It is designed to detect and measure the presence of the Estrogen Receptor alpha (ERα) protein in biological samples. The core function of this product is to provide researchers with a tool for analyzing the expression levels of this specific protein, which is important in various areas of biological and medical research.
Automatically generated - may contain errors
Lab products found in correlation
β actin ac 15, by Merck Group (2 mentions)
Anti mouse cy3, by Jackson ImmunoResearch (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Tissue culture plasticware, by BD (1 mentions)
β actin, by Merck Group (1 mentions)
Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Phospho akt s473 587f11, by Cell Signaling Technology (1 mentions)
Lmnb1, by Merck Group (1 mentions)
α β tubulin, by Cell Signaling Technology (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Vimentin 5741, by Cell Signaling Technology (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
A11037, by Thermo Fisher Scientific (1 mentions)
Bx40 microscope, by Olympus (1 mentions)
Phospho p44 42 mapk, by Cell Signaling Technology (1 mentions)
Phospho egfr, by Cell Signaling Technology (1 mentions)
Phospho erα, by Cell Signaling Technology (1 mentions)
Igf 1rβ, by Cell Signaling Technology (1 mentions)
4 protocols using erα sp1
1
Breast Cancer Cell Line Analysis
2
Monoclonal Antibody Generation against BIG3
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The anti-BIG3 monoclonal antibody was generated by Sigma-Aldrich (Tokyo, Japan). Briefly, a rat was immunised with purified His-tagged human BIG3 protein (459-572 aa). The iliac lymph nodes were collected and fused with myeloma cells, resulting in the formation of a hybridoma. Immunoblot analyses were performed as described previously9 (link). After SDS-PAGE, the membranes blotted with proteins were blocked with 4% BlockAce solution (Dainippon Pharmaceutical, Osaka, Japan) for 1 h and then incubated with antibodies against the following proteins: BIG3 (1:1,000); PHB2 (1:1,000, Abcam, Cambridge, UK); ERα (SP-1, 1:500, Thermo Fisher Scientific, Fremont, CA, USA); Akt, phospho-Akt (S473) (587F11, 1:1,000), p44/42 MAPK, phospho-p44/42 MAPK (T202/Y204) (1:1,000), VCP (valosin-containing protein) (1:500), and α/β-tubulin (1:1,000) (Cell Signaling Technology, Danvers, MA, USA); β-actin (AC-15; 1:5,000) and LMNB1 (1:100; Sigma) using standard procedures. All of the experiments were performed in triplicate at a minimum.
3
Immunocytochemistry for Breast Cancer Markers
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For immunocytochemistry (ICC), 1–2 × 105 cells were seeded onto glass coverslips in 6-well plates in regular media. For assessing PR, cells were treated with 10 nM E2 for 48 h prior to collection. Cells were washed twice with PBS and fixed with ice-cold 70% acetone/30% methanol for 5 min. Fixed cells were blocked with 10% normal goat serum (Vector Labs, Burlingame, CA) in 0.05% TBS-T for 30 min. Primary antibodies were as follows: ERα (SP1, 1:200, Thermo-Fisher, Waltham, MA), PR (1294, 1:50, [26 (link)], CK5 (ab75869, 1:400, Abcam, Cambridge, MA), CK8/18 (NCL-L-5D3, 1:2000, Leica Biosystems), and Vimentin (5741, 1100, Cell Signaling, Danvers, MA) for 1 h. Secondary antibodies were A11029 (green) and/or A11037 (red) (Invitrogen, 1:200) for 30 min followed by counterstaining with 0.1 μg/mL DAPI. Phase contrast images were captured using a Nikon TiE microscope (Nikon, Melville, NY) equipped with a digital camera and NIS Elements 4.6 software. Fluorescent images were captured using an Olympus BX40 microscope equipped with a digital camera and cellSens Standard 1.13 software. Adobe Photoshop CC 2019 was used to perform minimal linear adjustments to brightness/contrast and to assemble pictures into multipanel figures. Immunohistochemistry (IHC) of PDX was performed with the same antibodies for the indicated markers as previously described [21 (link)].
4
Molecular Analysis of Protein Signaling
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SDS-PAGE and immunoblot analyses were performed as described previously,21 (link) with antibodies against the following proteins: PHB2 (1:1000), ERα (phospho Y537; 1:500), ErbB2/HER2 and phosphor-HER2 (pY877) (Abcam, Cambridge, UK); ERα (SP-1, 1:500; Thermo Fisher Scientific, Fremont, CA, USA); Akt, phospho-Akt (S473) (587F11, 1:1000), p44/42 MAPK, phospho-p44/42 MAPK (T202/Y204) (1:1,000), Shc (1:500), phospho-Shc (Tyr239/240) (1H12, 1:500), phospho-ERα (S104/S106; 1:500), IGF-1Rβ (1:500), phospho-IGF-1Rβ (Y1135/1136) (19H7, 1:500), EGFR (1:1000) and phospho-EGFR (Tyr1068) (1:500) (Cell Signaling Technology); PI3-kinase p85α (U13; 1:500) and phospho-ERα (S118; 1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); phospho-ERα (S167) (1:500) and phospho-ERα (S305) (1:500) (Merck Millipore, Billerica, MA, USA); phosphotyrosine (1:500) (Life Technologies, Rockville, MD, USA); and β-actin (AC-15; 1:5000) (Sigma). All of the experiments were performed in triplicate at minimum.
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