Antibodies beta-actin (A2066; Sigma-Aldrich), cleaved Notch2 (C651.6DbHN; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), HES1 (ab71559; AbCam, Cambridge, MA; TA500014, Origene, Rockville, MD), Notch-1, −3, and-4(ab52627, ab23426, and ab184742, Abcam), Jagged1 (sc11376; Santa Cruz Biotechnology), DLL1 (sc73899; Santa Cruz Biotechnology), p53 (DO1; Santa Cruz Biotechnology), FITC anti-p53 antibody (645803; Biolegend, San Diego, CA), PLK1 (50-171-861; Millipore, Billerica, MA), CHFR (H00055743-M01; Abnova, Taipei, Taiwan), PAR (4335-MC-100-AC; Trevigen, Gaithersburg, MD), MDM2 (OP115; Millipore), pMDM2(ser260) (orb129684; Biorbyt LLC, San Francisco, CA) (Bioworld Technology, Inc., St Louis Park, MN), phycoerythrin-labeled donkey anti-rabbit IgG antibody (406421; Biolegend), and anti-ubiquitin (U5379; Sigma-Aldrich) were used. PARP1 inhibitor 3ABA (300 μM; Sigma-Aldrich) (27 (link)), dimethyl sulfoxide (DMSO), Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT; Sigma-Aldrich), and PLK1 inhibitors BI6727 (volasertib) (28 (link)) and BI2536 (29 (link)) (Selleck Chemicals, Houston, TX) were used.
Ab184742
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab184742 is an antibody product manufactured by Abcam. This product is intended for use in laboratory research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab23426, by Abcam (2 mentions)
Ab52627, by Abcam (2 mentions)
Ab8226, by Abcam (2 mentions)
Ab71559, by Abcam (1 mentions)
Par 4335 mc 100 ac, by Bio-Techne (1 mentions)
Anti ubiquitin, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
P53 do 1, by Santa Cruz Biotechnology (1 mentions)
Bi2536, by Selleck Chemicals (1 mentions)
Ab18976, by Abcam (1 mentions)
Ab668, by Abcam (1 mentions)
Mini hd9, by Uvitec (1 mentions)
Methanol activated polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Ab83232, by Abcam (1 mentions)
Ab59479, by Abcam (1 mentions)
Ab79559, by Abcam (1 mentions)
Ab8926, by Abcam (1 mentions)
7 protocols using ab184742
1
Investigating Notch Signaling Pathway
2
Western Blot Analysis of Stem Cell Markers
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Cells were lysed on ice and total protein was extracted using pre-cooled RIPA lysis buffer (P0013B, Beyotime) with protease inhibitors (Roche), followed by measurement of protein concentrations by BCA protein assay kit (CW00145, Cwbiotech, China). After being centrifuged, quantified and denatured, 80 μg protein samples were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and were then transferred onto methanol-activated polyvinylidene difluoride membranes (GE Healthcare, USA). Bovine serum albumin solutions (5%) were used to block non-specific antigens for 1.5 h. The membranes were probed with anti-Notch4 rabbit monoclonal antibody (1 : 500, ab184742, Abcam), anti-Oct4 rabbit monoclonal antibody (1 : 500, ab18976, Abcam), anti-Krt18 mouse monoclonal antibody (1 : 500, ab668, Abcam) or anti-GAPDH mouse monoclonal antibody (1 : 1000, 60 004–1-Ig, Proteintech). Protein bands were visualized using a UVITEC Alliance MINI HD9 instrument (UVITEC, UK) with an enhanced chemiluminescence detection system.
3
Western Blot Analysis of Cell Signaling
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Total cell lysates from cells were prepared using ProteoJET™ Mammalian Cell Lysis Reagent according to the manufacturer’s instructions (Thermo Scientific, Rockford, IL, USA), and protein concentrations were determined using the BCA method. Specific antibodies to JAG2 (ab109627), PRAF2 (ab53113), NICD (ab83232), E-cadherin (ab1416), vimentin (ab92547), CD63 (ab59479), CD81 (ab79559), NOTCH1 (ab52627), NOTCH2 (ab8926), NOTCH3 (ab23426), NOTCH4 (ab184742) and β-actin (ab8226) were purchased from Abcam (Cambridge, UK).
4
Protein Expression Analysis by Western Blot
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Equal amounts of protein samples were extracted from cells and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto a polyvinylidene fluoride membrane (Millipore, USA), blocked with 5% fat-free milk, then incubated with antibodies against ACE2 (1:3,000, ab108252, Abcam, US), VE-cadherin (1:1,000, ab33168, Abcam), EphA2 (1:1,000, 6997s, CST, USA), AKT (1:1,000, 4691, CST), p-AKT (1:2,000, 4060, CST), p38 (1:2,000, ab7952, Abcam), p-p38 (1:1,000, 4511, CST), Nodal (1:2,000, ab55676, Abcam), Notch4 (1:2,000, ab184742, Abcam), and Actin (1:2,000, ab8226, Abcam) overnight at 4°C, and the corresponding secondary antibodies (1:5,000, Thermo Fisher, USA) for 1 h at room temperature (RT). The blots were examined by an enhanced chemiluminescence detection kit (Millipore). Actin was used as a loading control, and all experiments were repeated three times independently. Expression quantification of proteins was performed using ImageJ software (National Institutes of Health, USA).
5
Notch Signaling Pathway Analysis
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Macrophages were homogenized in radioimmunoprecipitation lysis buffer (Thermo Scientific). Protein samples were obtained by centrifugation for 10 min at 14,000 g at 4°C. Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes by the wet blotting procedure (100 V for 2 h at 4°C). Membranes were blocked with blocking buffer (5% skimmed milk in PBS containing 0.05% Tween-20) and incubated at 4°C overnight with the anti-Notch1 (1:1000, #4380; Cell Signaling Technology), anti-Notch4 (1:1000, #ab184742; Abcam), anti-Hes1 (1:1000, #11988S; Cell Signaling Technology), anti-TGF-β (1:1000, #ab31013; Abcam), anti-Smad3 (1:1000, #9523S; Cell Signaling Technology), or anti-GAPDH (1:1000, #2118S; Cell Signaling Technology) primary antibody. GAPDH was used as an internal control. After incubation of the membrane with an IRDye 800 anti-rabbit or IRDye 680 anti-mouse secondary antibody (LI-COR, USA) for 1 h at room temperature, densitometric analysis was performed using an Odyssey infrared imaging system (LI-COR).
6
Immunohistochemical Analysis of Gastric Angiogenesis
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Gastric sections were embedded in paraffin and cut into 3 µm slices for IHC assay. The sections were heated at 97°C for 20 min, soaked in 3% hydrogen peroxide solution for 15 min and blocked with 5% bovine serum albumin for 30 min. The sections were incubated overnight at 4°C with primary antibodies against CD34 (lot ZDP0112111, R & D Systems, United States), VEGF-A (lot GR116031-1, Abcam, United Kingdom), HIF-1α (lot L1212, Santa Cruz Biotechnology, United States), Notch1 (ab52301, Abcam, United Kingdom), Notch4 (ab184742, Abcam, United Kingdom), and DLL4 (ab7280, Abcam, United Kingdom). Then, the sections were stained with diaminobenzidine and counterstained with hematoxylin to detect the results. Three fields were randomly selected under the light microscope for photographing. Quantification of expression levels was determined by mean of integrated optical density and analyzed by Image Pro Plus 6.0 software (Media Cybernetics, Inc.). The magnification used was ×200.
7
Quantifying Notch Receptor Expression in LUAD Cells
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Total proteins were obtained from LUAD cells with RIPA buffer (Solarbio, Shanghai, China) and quantified by BCA protein quantification kit (Solarbio, Shanghai, China). Approximately 40 µg of total protein were separated through a 10% SDS-PAGE and were then transferred to PVDF membranes. Membranes were next incubated with the specific primary antibody against Notch1 (1:1,000, ab280898; Abcam, Cambridge, MA, USA), Notch2 (1:3,000, ab245325; Abcam, Cambridge, MA, USA), Notch3 (1:600, ab252845; Abcam, Cambridge, MA, USA), Notch4 (1:600, ab184742; Abcam, Cambridge, MA, USA), and ubiquitin (1:1,500, ab134953; Abcam, Cambridge, MA, USA) for 1 h at room temperature (RT). After washing with TBST five times, membranes were incubated with HRP-coupled anti-rabbit or mouse secondary antibody (1:6,000) for 1 h at RT. Immunoblots were visualized with the chemiluminescence detection system.
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