Primary antibodies targeting Numb (1:1000 dilution; ab14140, Abcam, Cambridge, UK), NICD1 (1:1000 dilution; #3608, Cell Signaling, Danvers, MA, USA), NICD2 (1:5000 dilution; #5732, Cell Signaling, Danvers, MA, USA), NICD3 (1:500 dilution; ABP-PAB-10683, Allele Biotechnology, San Diego, CA, USA), NICD4 (1:1500 dilution; #2423, Cell Signaling, Danvers, MA, USA), E-cadherin (1:200 dilution; sc-7870, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Vimentin (1:200 dilution; V6630, Sigma-Aldrich, St. Louis, MO, USA), Snail (1:1000 dilution; #3879, Cell signaling, Danvers, MA, USA) were used. All analyses included staining with Ponceau S, which confirmed that protein loading was equal. Band intensity was demonstrated by quantitative densitometric analysis using National Institutes of Health (NIH) ImageJ Ver1.49 software (NIH, Bethesda, MD, USA). Standardization was performed by measuring actin in the same blots with an anti-actin antibody (1:1500 dilution; A2066, Sigma-Aldrich, St. Louis, MO, USA).
Nicd1
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
NICD1 is a protein that plays a key role in the Notch signaling pathway. It functions as the intracellular domain of the Notch receptor and is released upon Notch activation. NICD1 translocates to the nucleus and acts as a transcriptional regulator, modulating the expression of target genes involved in cell fate determination and differentiation.
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Lab products found in correlation
Snail, by Cell Signaling Technology (3 mentions)
Vimentin, by Merck Group (2 mentions)
Nicd2, by Cell Signaling Technology (2 mentions)
Nicd4, by Cell Signaling Technology (2 mentions)
E cadherin, by Santa Cruz Biotechnology (2 mentions)
Anti actin antibody, by Merck Group (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Notch1, by Cell Signaling Technology (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Ab14140, by Abcam (1 mentions)
H3k9me3, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg, by Promega (1 mentions)
Phosphor smad1 5 8, by Cell Signaling Technology (1 mentions)
Semi dry transfer apparatus, by Bio-Rad (1 mentions)
Cox 4, by Cell Signaling Technology (1 mentions)
Matrix metalloproteinase 9 mmp9, by Cell Signaling Technology (1 mentions)
2 2 2 trichloroethanol, by Merck Group (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
10 protocols using nicd1
1
Immunoblotting Analysis of Notch Pathway
2
ChIP Assay for Transcription Factor Binding
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ChIP assay was performed as previously described (41 (link)). Briefly, control and JMJD2D-knockdown cells were fixed with 1% formalin for 10 min, and then chomatin was immunoprecipitated using antibody for JMJD2D (Abcam, Cat# ab93694, 1:50), TCF4 (Cell Signaling Technology, Cat# 2569, 1:50), β-catenin (Cell Signaling Technology, Cat# 8480, 1:50), NICD1 (Cell Signaling Technology, Cat# 3608, 1:50), and H3K9me3 (Abcam, Cat# ab ab8898, 1:50). The ChIP DNA was isolated and detected by quantitative real-time PCR using specific EpCAM promoter primer (TCF4-binding element 1 (TBE1): 5′-TACATTAATCAACTTGCCGG-3′ and 5′-CAGAGCAAGACTCCATCTC-3′; TCF4-binding element 2 (TBE2): 5′-TCAACTTGCCGGCACTTCA-3′ and 5′-AGCAATTTTAAGATTCAGC-3′; and Sox9 promoter primer (5′-TAAGTCGGGAAGGTTCCTTG-3′ and 5′-GGCTTGCAGAATGCGGAACA-3′.
3
Western Blot Analysis of MSC Signaling
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MSCs cultured with or without OA and BMP2 treatment were lysed in RIPA buffer (10 mM Tris–HCl, 1 mM EDTA, 1% sodium dodecyl sulfate [SDS], 1% NP-40, 1: 100 proteinase inhibitor cocktail, 50 mM β-glycerophosphate, 50 mM sodium fluoride). The samples were separated on a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes with a semi-dry transfer apparatus (Bio-Rad). The membranes were blotted with 5% dehydrated milk for two hours, and then, incubated with primary antibodies overnight. The immune complexes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega, USA), and visualized with SuperSignal reagents (Pierce, USA). Primary polyclonal antibodies against NICD1 and phosphor-smad1/5/8 (Cell Signaling, USA) were used. We also used a primary monoclonal antibody to detect the housekeeping protein, β-actin (Sigma-Aldrich, USA).
4
Immunoblotting Assay Protocol
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Immunoblotting was performed as described (Ehrlich et al, 2015 (link)). The following primary antibodies were used: AKT (9272, Cell Signaling), AKT phospho (Ser 473; 4051, Cell Signaling), β-actin (MAB1501, Millipore), β-tubulin (2146, Cell Signaling), Bim (2819, Cell Signaling), Cdk5 (AHZ0492, Life Technologies), COX IV (4844, Cell Signaling), CREB (9104, Cell Signaling), ERK (9102, Cell Signaling), ERK phospho (9106, Cell Signaling), Foxo1 (2880, Cell Signaling), GAPDH (sc-69778, Santa Cruz), HIF1α (610958, BD Biosciences), phospho-histone H2aX (γH2aX, 2577, Cell Signaling), matrix metalloproteinase-9 (MMP-9; 3852 Cell Signaling), MMP-2 (4022 Cell Signaling), NICD-1 (4147, Cell Signaling), NICD-4 (sc-5594, Santa Cruz), Notch 1 (3608, Cell Signaling), Retinoblastoma protein phospho (Ser807/811; 8516, Cell Signaling), Retinoblastoma protein (554136, BD Biosciences), Stat3 phospho (Ser 727; 9134, Cell Signaling), Stat3 (9132, Cell Signaling). Anti-vimentin, -snail, -β-catenin, -claudin-1, N-cadherin were from Cell Signaling (EMT Sampler Kit, 9782), c-Myc (sc-788, Santa Cruz). Loading control by stain-free gels was performed by adding 0.5% TCE (2,2,2-Trichloroethanol, Sigma-Aldrich) to the PAGE gels according to the TGX Stain-Free Gels system (BioRad, Hercules, CA, USA).
5
KK-LC-1 Interaction with Presenilin-1
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Western blot was performed using an SDS‐PAGE Electrophoresis System as described previously20 with antibodies specific for KK‐LC‐1 (Abcam, Cambridge, MA, USA), E‐cadherin (Cell Signaling Technology, Beverly, MA, USA), N‐cadherin (Cell Signaling Technology), vimentin (Cell Signaling Technology), Snail (Cell Signaling Technology), NICD1 (Cell Signaling Technology), Hes1 (Cell Signaling Technology), presenilin‐1 (Abcam) and GAPDH (Cell Signaling Technology). Signals were detected using enhanced chemiluminescence reagents (Thermo Fisher Scientific). The total proteins were extracted from cells for co‐immunoprecipitation using protein A/G‐agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's instructions. Antibodies against KK‐LC‐1 (FabGennix, Frisco, TX, USA) and presenilin‐1 (Abcam) were used for co‐immunoprecipitation. The bound proteins were analysed by Western blot.
6
Quantitative Immunoblotting Analysis
7
Protein Expression Analysis in Cell Lines
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We used primary antibodies targeting DLL3 (1:750; ab103102; Abcam, Cambridge, UK), NOTCH1 intracellular domain (NICD1; 1:500; #3608; Cell Signaling Technology, Danvers, MA, USA), NICD2 (1:5000; #5732; Cell Signaling Technology), NICD3 (1:1000; 55114‐1‐AP; Proteintech, Rosemont, IL, USA), NICD4 (1:1500; #2423; Cell Signaling Technology), E‐cadherin (1:200; sc‐8426; Santa Cruz Biotechnology, Dallas, TX, USA), Vimentin (VIM; 1:200; V6630; Sigma‐Aldrich, St. Louis, MO, USA), Snail (1:1000; #3879; Cell Signaling Technology), phospho‐Smad2/Smad3 (1:1000; #8828; Cell Signaling Technology), Smad2/Smad3 (1:1000; #8685; Cell Signaling Technology), Smad4 (1:1000; #38454; Cell Signaling Technology) and ASCL1 (1:250; #556604; BD Pharmingen, Franklin Lakes, NJ, USA). All analyses included staining with Ponceau S, which confirmed equivalent protein loading. Standardization was performed by measuring actin with the anti–actin antibody (1:1500; A2066; Sigma‐Aldrich).
8
Notch Pathway Proteins Analysis
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Protein was isolated from Hs766T-L3 cells using lysis buffer (Beyotime Biotechnology, China) containing 1% protease inhibitors. Then denatured protein samples were subjected to SDS‐PAGE for electrophoresis. Next, the proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking in 5% skim milk at room temperature for 2 h, membranes were incubated with primary antibodies against NICD3 (1:1,000; Cell Signaling Technology), NICD1 (1:1,000; Cell Signaling Technology), and Actin (1:2,000; Santa Cruz Biotechnology) overnight at 4°C. After washing with TBST for three times, the membranes were incubated with appropriate secondary antibodies at 37°C for 2 h. High Sensitivity ECL kit was used to detect chemiluminescent signals.
9
Chidamide-Induced Apoptosis in Cancer
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DMSO was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chidamide was provided from the Chipscreen Company (Shenzhen, Guangzhou, China), dissolved in DMSO at a 100 mmol/L concentration. Fetal bovine serum was purchased from Sigma-Aldrich (St. Louis, MO, USA). MYC, cyclin D1, anti-histone H3, anti-histone H3 (acetyl K27) antibodies were purchased from Abcam (Cambridge, MA, USA); β-actin antibody was bought from Sigma-Aldrich; caspase-9, caspase-8, caspase-3, cleaved-caspase-3, PARP, cleaved PARP, Bcl-2, Bcl-xL, Bid, p21, mouse anti-rabbit IgG and NICD1 antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). CHX was purchased from Sigma, and MG132 was bought from Selleck (Shanghai, China).
10
Immunoblot Analysis of Protein Markers
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For immunoblot analyses, proteins were isolated using a Protein Extraction Kit (Beyotime Institute of Biotechnology, Jiangsu, China). Extracted protein was measured with the BCA protein assay reagent kit (Pierce, Madison, WI). Equal amounts of total protein (80 g of protein/lane) were resolved on 5-10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20 and incubated overnight at 4°C with primary antibodies against NICD1 (1:1,500; Cell Signaling, Beverly, MA), Hes1 (1: 800; Santa Cruz Biotechnology, Santa Cruz, CA), Arg1 (1:1,000; Abcam, Cambridge, MA), iNOS (1:500; Abcam), or NGF (1:1,000; Santa Cruz Biotechnology). The blots were developed using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA) and visualized using a FluorChem E Imager (Protein-Simple, Santa Clara, CA). The densities relative to GAPDH were analyzed using NIH ImageJ software.
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