Total protein was harvested from cells using RIPA lysis buffer (Beyotime, China). Protein samples were separated by sodium dodecyl-sulfate polyacrylamide (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membranes. Blots were washed and incubated with antibodies. The protein bands were then visualized with an ECL detection system. The primary and secondary antibodies utilized in this study were listed as follows: anti-GAPDH (60004-1-Ig, Proteintech, USA), anti-HNRNPAB Abcam, USA), anti-CDK1 (ab133327, Abcam, USA), anti-Cyclin B1 (ab32053, Abcam, USA), anti-CDC25A (sc-7389, Santa Cruz, USA), anti-CDC25C (ab32444, Abcam, USA), anti-ER (ab75635, Abcam, USA), anti-HER2 (ab134182, Abcam, USA), HRP-conjugated goat anti-mouse IgG (SA00001-1, Proteintech, USA), and HRP-conjugated goat anti-rabbit IgG (SA00001-2, Proteintech, USA).
Ab75635
Manufactured by Abcam
Sourced in United States
Ab75635 is a lab equipment product. It is a device used for measuring or analyzing samples in a laboratory setting. The core function of this product is to perform specific tasks related to scientific research or analysis, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (1 mentions)
Ab32053, by Abcam (1 mentions)
Ab133327, by Abcam (1 mentions)
Ab32444, by Abcam (1 mentions)
Ab134182, by Abcam (1 mentions)
Cdc25a sc 7389, by Santa Cruz Biotechnology (1 mentions)
Sa00001 1, by Proteintech (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Odyssey system, by LI COR (1 mentions)
Nupage reducing agent, by Thermo Fisher Scientific (1 mentions)
Ab3576, by Abcam (1 mentions)
Ab74272, by Abcam (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (1 mentions)
Biotin blocking buffer, by Vector Laboratories (1 mentions)
G8796, by Merck Group (1 mentions)
Fusion fx, by Vilber (1 mentions)
Quick start bradford protein assay, by Bio-Rad (1 mentions)
9 protocols using ab75635
1
Western Blot Quantification of Proteins
2
Western Blot Analysis of Astrocyte and Neuron Markers
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Expression of ERα, ERβ, GFAP, cleaved-Caspase-3 and Caspase-3 protein in primary cultured astrocytes, neurons or hippocampus were measured by western blot. In brief, soluble lysates of samples were mixed with sample buffer and NuPAGE reducing agent (Invitrogen). The extracted proteins were separated using 10% SDS-PAGE and then electrically transferred to polyvinylidene difluoride membranes. Subsequently, the membranes were blocked in 5% nonfat dry milk diluted in TBST for 1 h at room temperature. The western blots were probed with rabbit anti-ERα antibody53 (link) (ab75635, 1:500, Abcam), rabbit anti-ERβ antibody54 (link) (ab3576, 1:500, Abcam), mouse anti-GFAP antibody (GA5, 1:1000, Cell Signaling Technology), rabbit anti-cleaved-Caspase-355 (link) (#9661, 1:1000, Cell Signaling Technology) antibody, rabbit anti-Caspase-3 antibody56 (link) (#9662, 1:1000, Cell Signaling Technology) and mouse β-actin antibody57 (link) (#3700, 1:1000, Cell Signaling Technology) overnight at 4 °C. The membranes were then incubated with an IRDye secondary anti-rabbit and anti-mouse antibody (Thermo Scientific, USA) for 2 h. Protein bands were visualized using the LI-COR Odyssey System (LI-COR Biotechnology, USA).
3
Immunohistochemical Analysis of ER and AR
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All tissue sections (4-µm thick) were processed for IHC as previously described (18 (link),19 (link)). The following primary antibodies were used for IHC: Anti-ERα (cat. no. ab75635; 1:200; Abcam) and AR (cat. no. ab74272; 1:250; Abcam).
The expression of ERα and AR was evaluated in terms of staining intensity: (Negative), 1 (weak), 2 (medium) or 3 (strong). The extent of staining was scored as 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%) or 4 (76-100%), according to the percentage of positively stained areas in relation to the whole tumor area. The final staining score (0-12) was calculated by multiplying the intensity score and the extent score (19 (link)). The final staining score was used to evaluate the expression of ERα and AR.
The expression of ERα and AR was evaluated in terms of staining intensity: (Negative), 1 (weak), 2 (medium) or 3 (strong). The extent of staining was scored as 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%) or 4 (76-100%), according to the percentage of positively stained areas in relation to the whole tumor area. The final staining score (0-12) was calculated by multiplying the intensity score and the extent score (19 (link)). The final staining score was used to evaluate the expression of ERα and AR.
4
Western Blot Analysis of ER-Alpha Expression
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The total proteins of the MCF-7/ADM and the MCF-7/WT cells were extracted using the CytoBuster™ Protein Extraction Reagent (Novagen, Madison, WI, USA) at 4°C. Subsequently, the proteins were resolved by 10% SDS-PAGE and transferred electrophoretically (at 100 V, for 1 h) onto polyvinylidene difluoride membranes, which were blocked (for 1 h, at 20°C) with 3% bovine serum albumin (BSA). The membranes were then incubated with the primary antibody, anti-human estrogen receptor alpha (ab75635, 1:2,000 dilution; Abcam, Cambridge, MA, USA) overnight at 4°C. Anti-GAPDH (ab9483, 1:1,000 dilution; Abcam) was used as a loading control. After incubation with the proper secondary antibodies, antibody binding was detected with an Odyssey Imaging system (LI-COR Biosciences, Lincoln, NE, USA).
5
Immunofluorescent Analysis of Estrogen Signaling in hESCs
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HESCs were cultured in 25 mm2 dishes and treated with E2 (Sigma–Aldrich) at 10−8 mol/l for 48 h, with DMSO as a control. Then, the cells were fixed in ice-cold 4% paraformaldehyde for 10 min, washed with 1× PBS, and permeabilized in PBS containing 0.5% Triton X-100 for 3 min at RT. All cells were blocked (5% BSA, 30 min, RT) and incubated overnight (4 °C) with primary antibodies directed against β-catenin (#8814, Cell Signaling Technology), LEF1 (#2286, Cell Signaling Technology), TCF3 (#2883, Cell Signaling Technology), and ESR1 (ab75635, Abcam). After the cells were exposed to the primary antibodies, they were incubated with Alexa Fluor 488- and 568-conjugated secondary antibodies and mounted with Prolong Gold antifade medium. Immunofluorescently labeled cell monolayers were visualized under an Olympus FV1000 laser scanning confocal microscope (Olympus).
6
Immunohistochemical Detection of ERα
7
Quantifying Estrogen Receptor Signaling
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ER protein abundance was assessed by immunoblot analysis using antibodies against ERα (ab75635, Abcam), ERβ (26 (link)), and GAPDH (G8796, Sigma-Aldrich) as a control. Phosphorylation of ERK1/2, p38 MAPK, and Akt proteins were assessed by immunoblot with their phosphorylated protein-specific antibodies (Cell Signaling), and their total protein abundance was also assessed.
8
Protein Extraction and Quantification from Frozen Tissue
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Frozen cardiac tissue was processed using a tissue homogenizer in RIPA Lysis Buffer System® (Life Technologie, Zug, Switzerland). Quick Start™ Bradford Protein Assay (Bio-Rad Laboratories, Cressier, France) was used for protein lysate concentration measurements. Rabbit anti-estrogen receptor α primary antibodies (1:1000, ab75635, Abcam, Cambridge, UK) and mouse anti-GAPDH (1:40,000, mab374, Merck, Schaffhausen, Switzerland) were measured. Membranes were imaged using FusionFX (Vilber, Marne-la-Vallée, France).
9
Co-Immunoprecipitation of β-Catenin and ER-α
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For co-immunoprecipitation (COIP) assays, cells were lysed in 400 μl lysis buffer (20 mM Tris (pH 7.4), 50 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.5% SDS, 0.5% deoxycholate, and protease inhibitors). Lysate aliquots (500 μg at 1 μg/μl) were precleared with 60 μl protein A-sepharose beads (Beyotime, Haimen, China) for 1 h at 4 °C. Appropriate amounts of mouse-directed antibody against β-catenin (sc-133239, Santa Cruz Biotechnology), mouse antibody directed against estrogen receptor alpha (ESR1; sc-73479, Santa Cruz Biotechnology), and mouse nonspecific IgG (Biosense, Bergen, Norway) were then added and incubated for 4 h at 4 °C. Preblocked agarose beads (100 μl) were added to the antibody/lysate mixture and incubated overnight at 4 °C. After the beads were washed three times with 1× PBS, the bound proteins were eluted in SDS sample buffer, resolved by SDS–PAGE, and analyzed by immunoblotting. The antibodies used for immunoblotting were rabbit directed antibodies against ESR1 (ab75635, Abcam), β-catenin (#8814, Cell Signaling Technology), LEF1 (#2286, Cell Signaling Technology), and TCF3 (#2883, Cell Signaling Technology).
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