Protein concentrations were determined with a BCA protein assay kit (Beyotime, P0012i). Twenty to thirty micrograms of total protein was separated by 10% SDS-PAGE (EpiZyme, pg112) and then transferred onto nitrocellulose membranes (Pall, 66485). Nonspecific sites were blocked with 5% nonfat milk in phosphate-buffered saline (with 0.1% Tween-20) at room temperature. Next, the blots were incubated overnight at 4 ℃ with the following primary antibodies: anti-Hbα (Santa Cruz, sc-514378), anti-Hbβ (Santa Cruz, sc-21757), anti-CD31 (Affinity, AF6191), anti-glutathione peroxidase 4 (GPX4) (Affinity, DF6701), anti-heme oxygenase-1 (HO-1) (Affinity, AF5393), anti-Bcl2 (ImmunoWay, YM3041), anti-Bax (ImmunoWay, YT0455), anti-Notch1 (Cell Signaling Technology, #3608), anti-Jag1 (Cell Signaling Technology, #70109), anti-Hes1 (Abcam, ab108937), anti-Hey1 (Abcam, ab154077), and anti-β-actin (Affinity, AF7018). The next day, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Affinity, s0001) and visualized with New Super ECL assay kit (KeyGen BioTECH, KGP1128).
Anti hey1
Manufactured by Abcam
Sourced in United States
Anti-Hey1 is a primary antibody that binds to the Hey1 protein. Hey1 is a transcriptional repressor that plays a role in various cellular processes. The antibody can be used for applications such as Western blotting, immunohistochemistry, and immunocytochemistry to detect and study the Hey1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti hes1, by Abcam (2 mentions)
Anti notch1, by Cell Signaling Technology (2 mentions)
Anti β actin, by Affinity Biosciences (2 mentions)
Anti hes1, by Cell Signaling Technology (2 mentions)
Anti bax, by Immunoway (1 mentions)
Anti jag1, by Cell Signaling Technology (1 mentions)
Anti cd31, by Affinity Biosciences (1 mentions)
New super ecl assay kit, by Keygen Biotech (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti bcl 2, by Immunoway (1 mentions)
Anti notch1, by Abcam (1 mentions)
Blocking buffer, by Biosharp (1 mentions)
Ripa containing protease inhibitors, by Beyotime (1 mentions)
Enhanced bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Anti proliferating cell nuclear antigen, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Anti mmp2, by Abcam (1 mentions)
Anti mmp9, by Abcam (1 mentions)
Super ecl detection reagent, by Yeasen (1 mentions)
Anti runx3, by Abcam (1 mentions)
6 protocols using anti hey1
1
Western Blot Analysis of Protein Markers
2
Western Blot Analysis of Notch Signaling Pathway
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Tissues or cells were lysed with RIPA containing protease inhibitors (Beyotime, China) on ice for 30 min. Samples were separated on SDS-PAGE gels and blotted onto PVDF membranes. After being blocked with blocking buffer (Biosharp, China) for 1 hour, the PVDF membranes were incubated with the following diluted primary antibodies at 4°C overnight: anti-CENPU (Affinity, China, 1 : 1000), anti-Notch1 (Abcam, USA, 1 : 1000), anti-Hes1 (Abcam, USA, 1 : 1000), anti-Hey1 (Abcam, China, 1 : 1000), and anti-β-actin (Affinity, China, 1 : 2000). Then, the PVDF membranes were incubated with specific horseradish peroxidase- (HRP-) conjugated IgG secondary antibodies (Affinity, 1 : 2000). The protein bands on the PVDF membrane were visualized by using a Tanon 5200 Imaging System.
3
Protein Expression Analysis in HTR-8/SVneo Cells
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Protein samples were isolated from HTR-8/SVneo cells using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.), quantified by an Enhanced Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology) and separated using 10% SDS-PAGE (30 µg per lane) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk powder in Tris-buffered saline containing 0.1% Tween-20 for 2 h at room temperature, followed by incubation with the following primary antibodies overnight at 4˚C: Anti-PSMD14 (1:1,000; cat. no. ab182762; Abcam), anti-nuclear protein Ki67 (1:1,000; cat. no. ab15580; Abcam), anti-proliferating cell nuclear antigen (PCNA; 1:1,000; cat. no. ab29; Abcam), anti-MMP2 (1:1,000; cat. no. ab92536; Abcam), anti-MMP9 (1:1,000; cat. no. ab76003; Abcam) and anti-HEY1 (1:1,000; cat. no. ab154077; Abcam). The secondary antibodies, goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (1:3,000; cat. no. ab6721; Abcam) and goat anti-mouse horseradish peroxidase-conjugated IgG secondary antibody (1:3,000; cat. no. ab6728; Abcam), were incubated at room temperature for 2 h. Super ECL Detection Reagent (Shanghai Yeasen Biotechnology Co., Ltd.) and ImageJ software v1.8.0 (National Institutes of Health) were used to observe and analyze the blots.
4
Immunohistochemical and Immunoblotting Analysis of Tumor Markers
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Formaldehyde- or paraformaldehyde-fixed tumor tissues were embedded in paraffin, and sections were stained with H&E using standard techniques. The following primary antibodies were used: anti-FLAG M2 (Sigma-Aldrich), anti-Sox9 (Sigma-Aldrich), anti-Hey1 (Abcam), anti-Runx2 (MBL), anti-CK18 (Proteintech), anti-Galectin 3 (Proteintech), anti-Ki-67 (Abcam), and anti-NCOA2/SRC2 (Cell Signaling).
Immunoblotting was performed using whole-cell lysates. The following primary antibodies were used: anti-FLAG M2 (Sigma-Aldrich), anti-Runx2 (MBL), anti-Runx3 (Abcam), anti–α-tubulin (Sigma-Aldrich), and anti-Cas9 (Novus, Centennial, CO).
Immunoblotting was performed using whole-cell lysates. The following primary antibodies were used: anti-FLAG M2 (Sigma-Aldrich), anti-Runx2 (MBL), anti-Runx3 (Abcam), anti–α-tubulin (Sigma-Aldrich), and anti-Cas9 (Novus, Centennial, CO).
5
Western Blot Analysis of Notch Signaling
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Cell lysate was used to treat cells and tissues. The protease inhibitor PMSF (Beyotime) was added. The BCA method was used to measure the protein concentration. Proteins were extracted by SDS‒PAGE and then transferred to nitrocellulose membranes. The nitrocellulose membranes were blocked with 5% skimmed milk prepared with TBST and subsequently incubated with the diluted primary antibodies, namely, anti-Notch1 (1:1000), anti-Vimentin (1:1000), anti-N-cadherin (1:1000), anti-E-cadherin (1:1000), anti-NICD (1:1000), and anti-HES1 (1:1000) (Cell Signaling Technology, CST, Boston, USA), anti-HEY1(1:1000) (Abcam, Cambridge, UK) at 4 °C overnight. After TBST was eluted on three occasions, horseradish peroxidase-labeled anti-rabbit or anti-mouse secondary antibodies (CST) were added and incubated on a shaking table for 1 h at room temperature. Then, the target bands were imaged using a chemiluminescence detection system (EMD, Millipore, Billerica, MA, USA) and analyzed quantitatively by using ImageJ software (National Institutes of Health, Bethesda, MD, USA). β-actin (1:5000) was used as an internal reference.
6
Notch Pathway Inhibition in Cancer
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5-FU, dimethyl sulfoxide (DMSO), and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine-t-butyl ester (DAPT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). αM was provided by professor SY Seo (College of Pharmacy, Gachon University, Republic of Korea) (Fig. 1 A). 5-FU and αM were dissolved in DMSO. The following antibodies were used for Western blotting and flow cytometry: anti-β-actin (1:1000, Gene Tex, Irvine, USA), anti-HES1 (1:1500, Cell Signaling, Danvers, MA, USA), anti-Notch1, anti-NICD 1 (1:100, Santa Cruz, TX, USA), anti-Hey1 (1:500, abcam, Cambridge, UK), fluorescein (FITC)-conjugated anti-CD44 (1:20, BD bioscience, Franklin Lakes, NJ), and phycoerythrin (PE)-conjugated anti-CD133 (1:50, Miltenyi Biotec, Bergisch Gladbach, Germany).
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