Protein lysates in NuPAGE buffer (Invitrogen) were separated by gradient 4–12% BisTris Gel and transferred to polyvinylidene difluoride membrane. Primary antibodies used in these experiments included rabbit monoclonal anti-Notch1 (D1E11; 1:1000; Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-cleaved Notch1 (Val1744; 1:1000; Cell Signaling), rabbit monoclonal anti-Notch2 (8A1; 1:1000; Cell Signaling), rabbit polyclonal anti-Notch3 (Pro2311; 1:1000; Cell Signaling), rabbit polyclonal anti-HES1 (ab71559; 1:1000; Abcam, Cambridge, MA, USA), rabbit polyclonal anti-HEY1 (ab22614; 1:500; Abcam), rabbit polyclonal anti-HES6 (ab66461; 1:1000; Abcam) and mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (clone 6C5; 1:1000; Santa Cruz Biotechnology, Dallas, Tx, USA). The membrane was incubated sequentially with primary antibodies (overnight at 4°C), horseradish peroxidase-conjugated anti-rabbit or antimouse secondary antibodies, and chemiluminescent substrate (Thermo Fisher, Waltham, MA, USA) before exposure to film.
Ab22614
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab22614 is a lab equipment product offered by Abcam. It is designed for laboratory use, but a detailed description of its core function is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab71559, by Abcam (2 mentions)
Ab25404, by Abcam (2 mentions)
Ab52627, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab66461, by Abcam (1 mentions)
Val1744, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Nupage buffer, by Thermo Fisher Scientific (1 mentions)
Ab7977, by Abcam (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ab32370, by Abcam (1 mentions)
Ab7280, by Abcam (1 mentions)
Ab39256, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
Anti ephrin b2, by Santa Cruz Biotechnology (1 mentions)
Anti ephb4, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Ab23426, by Abcam (1 mentions)
11 protocols using ab22614
1
Notch Signaling Pathway Protein Analysis
2
Western Blot Analysis of Cellular Proteins
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Proteins from vessels (12–15 µg) were resolved on SDS-PAGE (12% resolving, 5% stacking) prior to transfer onto nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). Membranes were stained with Ponceau S and probed for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to ensure equal protein loading and transfer and rinsed in wash buffer (PBS containing 0.05% Tween-20) before being probed as described previously [29] (link). Antibodies were purchased from Abcam for Notch 1 (ab52627), Hrt-1 (ab22614), Hrt-2 (ab25404), Bax (ab7977), Bcl-XL (ab32370) and PCNA (ab29).
3
Shear Stress-Induced Protein Expression
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Cells grown in glass slides were treated with shear stress for 2 h. Then total protein was obtained by using M-PER protein extraction buffer, and quantified using a BCA kit (Thermo Fisher Scientific, Inc.) and separated on 7.5 or 12% polyacrylamide gel followed by transfer onto an ImmunBlot PVDF membrane (GE Healthcare Life Sciences, Little Chalfont, UK). The membranes were blocked for 1 h with 5% BSA in Tris-phosphate buffer containing 0.05% Tween-20 (TBS-T). It was further incubated overnight at 4°C with anti-Notch1 (1:2,000; ab52627), anti-DLL4 (1:1,000; ab7280), anti-Hey1 (2 μg/ml, ab22614), anti-Hey2 (2 μg/ml; ab25404) all from Abcam (MA, USA); anti-EphrinB2 (1:1,000; sc-398735) and anti-EphB4 (1:1,000; sc-130081) both from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); anti-VEGFR2 (1:2,000; ab39256) and anti-CD31 (1:500; ab28364) both from Abcam, primary antibodies. After three washes (5 min) with TBS-T, membranes were further incubated with HRP-conjugated secondary antibodies: anti-rabbit (cat. no. 7074) or anti-mouse (cat. no. 7076) both from Cell Signaling Technology, Inc., (Danvers, MA, USA) for 1-2 h and followed by three washes with TBS-T. The target protein signal was detected and digitized using ECL (Thermo Fisher Scientific, Inc.).
4
Niclosamide Induces Notch Pathway Inhibition
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Niclosamide was obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; also known as thiazolyl blue and methylthiazolyldiphenyl tetrazolium bromide) was purchased from Sigma-Aldrich. The Annexin V-FITC Apoptosis Detection kit was purchased from Vazyme Biotech (Nanjing, China). Dimethyl sulfoxide (DMSO) was obtained from BioSharp (Hefei, China). The primary antibodies, rabbit polyclonal anti-Notch1 (ab27526), rabbit polyclonal anti-Notch2 (ab8926), rabbit polyclonal to anti-Notch3 (ab23426) and rabbit polyclonal anti-Hey1 (ab22614) were purchased from Abcam (Cambridge, UK). Mouse anti-β-actin monoclonal antibody (TA-09) was purchased from ZSGB-BIO (Beijing, China). The secondary antibodies, peroxidase-conjugated AffiniPure goat anti-rabbit IgG (ZB-2301) and peroxidase-conjugated AffiniPure goat anti-mouse IgG (ZB-2305), were purchased from ZSGB-BIO.
5
Western Blot Analysis of Cell Signaling
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Total proteins were isolated from transfected and/or treated cells through using the RIPA buffer (Beyotime, Shanghai, China), and quantified with a bicinchoninic acid (BCA) protein quantification kit. Then, approximately 30 μg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes. Subsequently, membranes were interacted with primary antibodies against p53 (1:1000, ab131442, Abcam, Cambridge, MA, USA), CyclinD1 (1:20,000, ab134175, Abcam), Pro-caspase-3 (1:1000, ab32150, Abcam), Cleaved-caspase-3 (1:1000, ab2302, Abcam), Pro-caspase 9 (1:10,000, ab32539, Abcam), Cleaved-caspase-9 (1:1000, ab2324, Abcam), HEY1 (1:5000, ab22614, Abcam), glyceraldehyde 3-phosphate dehydrogenase (GADPH) (1:10,000, ab8245, Abcam) and the secondary HRP-conjugated antibody (1:5000, ab20272, Abcam). Finally, protein bands were detected using electrochemiluminescence.
6
Immunofluorescence Staining of ADAMTS1, Dll4, HEY1, and Jagged1
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The primary
antibodies used in this study were raised against ADAMTS1
(3C8F4) mouse mAb IgG1k, sc-47 727 (Santa Cruz Biotechnology,
Dallas, TX); Dll4 (G-12) mouse mAb IgG2a, sc-365 429 (Santa
Cruz Biotechnology, Dallas, TX); HEY1 rabbit pAb, ab22614 (Abcam,
Cambridge, UK); and Jagged1 rabbit pAb, ab7771 (Abcam, Cambridge,
UK). The following secondary antibodies were used for this study:
Alexa Fluor 488-conjugated goat antimouse IgG (H + L), A-11 001 and
Alexa Fluor 647-conjugated chicken antirabbit IgG (H + L), A-21 443.
Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) was applied in a dilution
of 1:100 with an end concentration of 5 μg/mL. The FluorSave
Reagent mounting medium was purchased from Merck Millipore (Darmstadt,
Germany).
antibodies used in this study were raised against ADAMTS1
(3C8F4) mouse mAb IgG1k, sc-47 727 (Santa Cruz Biotechnology,
Dallas, TX); Dll4 (G-12) mouse mAb IgG2a, sc-365 429 (Santa
Cruz Biotechnology, Dallas, TX); HEY1 rabbit pAb, ab22614 (Abcam,
Cambridge, UK); and Jagged1 rabbit pAb, ab7771 (Abcam, Cambridge,
UK). The following secondary antibodies were used for this study:
Alexa Fluor 488-conjugated goat antimouse IgG (H + L), A-11 001 and
Alexa Fluor 647-conjugated chicken antirabbit IgG (H + L), A-21 443.
Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) was applied in a dilution
of 1:100 with an end concentration of 5 μg/mL. The FluorSave
Reagent mounting medium was purchased from Merck Millipore (Darmstadt,
Germany).
7
Immunoblotting Analysis of HEY1 Protein
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Cells were lysed by using NP40 lysis buffer (0.5% NP40, 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 3 mM pAPMSF (Wako Chemicals, Kanagawa, Japan), 5 mg ml−1 aprotinin (Sigma, St. Louis, MO, USA), 2 mM sodium orthovanadate (Wako Chemicals) and 5 mM EDTA. Lysates were subjected to SDS-PAGE followed by immunoblotting. The following antibodies were used: anti-HEY1 (1 : 200, ab22614, Abcam, Cambridge, UK; Figure 1B , Supplementary Figure 2B ), anti HEY1 (1 : 200, H00023462-M01, Abnova, Taipei, Taiwan; Figure 3) and β-tubulin (1 : 1000, DM1A, Sigma-Aldrich Co. LLC). Detection was performed by using the enhanced chemiluminescence detection system (Amersham, Giles, UK).
8
Protein Expression Analysis in Cells
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Cells were transfected and/or treated and then lysed in lysis buffer (Beyotime, Shanghai, China). For nucleus protein isolation, cells were lysed by Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) according to the manufacture's instruction. The cell protein extracts (30 ~ 50 µg) were separated by sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) electrophoretically transferred onto polyvinylidene difluoride membranes, which were then blocked with 5% non‐fat dried milk in TBS‐T and then incubated with proper primary antibodies listed below: anti‐α‐SMA (14395‐1‐AP; ProteinTech, Rosemont, IL, USA), anti‐vimentin (60330‐1‐lg; ProteinTech), anti‐E‐cadherin (20874‐1‐AP; ProteinTech), anti‐Notch1 (ab83232; Abcam), anti‐C‐MYC (ab32072; Abcam), anti‐HES1 (ab71559; Abcam), anti‐HEY1 (ab22614; Abcam), anti‐GAPDH (60004‐1‐lg, ProteinTech, USA), anti‐Histone H3(17168‐1‐AP, ProteinTech) and anti‐β‐actin (60008‐1‐lg, ProteinTech). After washing with TBST buffer, the membranes were incubated with the appropriate secondary antibodies. Immunoreactive proteins were visualized using enhanced chemiluminescence (ECL) techniques.
9
Protein Extraction and Western Blot Analysis
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Proteins were extracted using the RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and Complete Lysis-M reagent (Roche, USA) followed by determination of their concentrations using the BCA assay (ThermoFisher Scientific, Waltham, MA). The proteins were resolved on SDS-PAGE gels (8%–10%), then transferred onto PVDF membranes for detection. We procured the following antibodies NOTCH1 (1:1000, 4380T, CST), HES1(1:1000, 11988S, CST), HEY1(1:1000, ab22614, Abcam), Bax (1:2000, ab182733, Abcam), Bcl-2(1:1000, ab32124, Abcam), E-cadherin(1:1000, 5296S, CST), Vimentin(1:1000, 5741T, CST), GAPDH (1:1000, ab9484, Abcam), from Cell Signaling Technology and Abcam (China) for use in the study.
10
Immunofluorescence Analysis of ALDH1A1 and Hey1 in Notch+ and Notch- Cells
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