Cytosolic protein was isolated from cells in lysis buffer A, containing Hepes (10 mM), KCl (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), DTT (1 mM), PMSF (0.5 mM), Protease Inhibitor Cocktail (15 µL/mL; Sigma-Aldrich), Igepal CA-630 (0.5%; Sigma-Aldrich), pH 7.9. The suspension was centrifuged, and the supernatant (cytosolic extract) was stored. Nuclear extracts were obtained by incubating the pellet obtained from the cytosolic extract in lysis buffer B, containing HEPES (20 mM), NaCl (0.4 M), EDTA (1 mM), EGTA (1 mM), DTT (1 mM), PMSF (0.5 mM), Protease Inhibitor Cocktail (15 µL/mL), pH 7.9. Protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). To determine specific protein contents, 20 µg of nuclear cell lysates were analyzed by immunoblotting using antibodies against proliferating cell nuclear antigen (PCNA; 1:100; Santa Cruz Biotechnology INC, Dallas, TX, USA), Cyclin D1 (1:500; Cell Signaling Technology Inc., Danvers, MA, USA) and cleaved Notch 1 (Val1744) (1:200; Cell Signaling Technology) as primary antibodies, and horseradish peroxidase-conjugated goat anti-mouse (1:10000; Santa Cruz Biotechnology) and goat anti-rabbit (1:10000; Santa Cruz Biotechnology) as secondary antibodies. Transcription factor II B (TFIIB; 1:1000; Cell Signaling Technology) was used as loading control.
Cleaved notch1 val1744
Manufactured by Cell Signaling Technology
Sourced in United States
Cleaved Notch1 (Val1744) is a lab equipment product offered by Cell Signaling Technology. It is an antibody that specifically detects the cleaved form of the Notch1 protein, which plays a key role in cell-cell communication and developmental processes. The product is intended for research use only.
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Lab products found in correlation
β actin, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bradford assay, by Bio-Rad (2 mentions)
C myc, by Santa Cruz Biotechnology (2 mentions)
Notch1, by Abcam (2 mentions)
Ab15592, by Abcam (2 mentions)
Ab109429, by Abcam (2 mentions)
Ab18189, by Abcam (2 mentions)
Ps1 ntf, by Merck Group (2 mentions)
Ps1 ctf, by Merck Group (2 mentions)
Ps2 ctf, by Abcam (2 mentions)
Aph 1al, by Thermo Fisher Scientific (2 mentions)
Anti fragilis, by Abcam (2 mentions)
Rab7 b 3, by Santa Cruz Biotechnology (2 mentions)
Imagej, by LI COR (2 mentions)
β actin c4, by Santa Cruz Biotechnology (2 mentions)
Image studio lite, by LI COR (2 mentions)
Immobilon fl pvdf, by Merck Group (2 mentions)
C myc, by Roche (2 mentions)
Immobilon p pvdf, by Merck Group (2 mentions)
18 protocols using cleaved notch1 val1744
1
Analysis of Nuclear Protein Markers
2
Interplay between ADAMTS1 and Notch1 in cell signaling
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HEK-293T (ATCC) cells were co-transfected with ADAMTS1 and Notch1 expression plasmids. The co-immunoprecipitation was performed using magnetic dynabeads protein G (Invitrogen). In co-culture experiments, HEK-293T cells expressing NOTCH1 and ADAMTS1 were co-cultured with NIH-3T3 cells (ATCC) expressing DLL1 for 5 h. The cell lysate were analyzed by co-immunoprecipitation and western blot. For western blotting, the muscle tissues and cells were extracted in RIPA lysis buffer (1× PBS, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA). Protein extracts were subjected to electrophoresis on polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were first incubated in blocking buffer and then incubated with antibodies. The antibodies used in this study were against: β-actin (Sigma, 1:5000, clone C2), NOTCH1 (BD Pharmingen, 1:200, clone MN1A), cleaved NOTCH1 val 1744 (Cell Signaling, 1:1000, clone 2421S), V5 (1:1000, Invitrogen) and ADAMTS1 (R&D Systems, 1:1000, clone CDSL0115031). Uncropped immunoblots are located in Supplementary Fig. 5 .
3
Western Blot Analysis of Cleaved Notch1, c-Myc, p19Arf, and p-p53
4
Protein Expression Analysis by Western Blotting
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Protein extractions and western blotting were performed as described previously [30 (link)]. Primary antibodies include antibodies against Tspan5 (1 : 3000, SAB2108599, Sigma‐Aldrich, St. Louis, MO, USA), Flag‐tag (1 : 1000, F1804, Sigma‐Aldrich), E‐cadherin (1 : 1000, BF0219, Affinity Biosciences, Cincinnati, OH, USA), N‐cadherin (1 : 1000, AF4039, Affinity Biosciences), vimentin (1 : 1000, #5741, Cell Signaling Technology, Boston, MA, USA), Snail (1 : 1000, #3895, Cell Signaling Technology), ADAM10 (1 : 1000, #14194, Cell Signaling Technology), cleaved Notch1 (Val1744) (1 : 1000, #4147, Cell Signaling Technology), Hes5 (1 : 1000, ab194111, Abcam, Cambridge Biomedical Campus, Cambridge, UK) and GAPDH (1 : 5000, Bioworld Technology, Bloomington, MN, USA). GAPDH was used as a loading control to normalize the protein expression. Protein bands on the blots were visualized by ECL chemiluminescence reagent and quantified by imagej software (NIH, Bethesda, MD, USA) for densitometry analysis. Each experiment was repeated at least three times.
5
Western Blot Analysis of Notch Signaling
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MYO10 (Novus Biologicals, NBP1-87748) was used at 1:2000. JAG1 (Cell Signaling Technology, 70109) was used at 1:2000. IL13RA2 (Abcam, ab55275) was used at 1:2000. Cleaved Notch1 (Val1744) (Cell Signaling Technology, 4147) was used at 1:2000. α-Tubulin (Millipore, MAB1864) was used at 1:5000. Actin (Sigma, A2066) was used at 1:5000. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, 2118) was used at 1:5000. Peroxidase AffiniPure Goat Anti-Mouse immunoglobulin G (IgG) (H+L) (Jackson ImmunoResearch, 115-035-003) was used at 1:10,000. Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, 111-035-144) was used at 1:10,000. Peroxidase AffiniPure Goat Anti-Rat IgG (H+L) (Jackson ImmunoResearch, 112-035-003) was used at 1:10,000.
6
Notch1 Activation and Immunoprecipitation
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Cells were seeded onto Jagged1-coated plates to activate Notch signaling for 24 h in DMEM-low glucose supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were lysed with 1 mL of ionic buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% NP40, 0.5% sodium deoxycholate, and 0.05% SDS) supplemented with protease inhibitor cocktail, phosphatase inhibitor, and 10 μM MG132 (all from Roche). Protein A/G Plus-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and cleaved Notch1 (Val 1744; Cell Signaling Technology) were used for immunoprecipitation. Agarose beads were washed several times with cold Tris buffer containing 1 mM Tris, pH 7.4, and 15 mM NaCl (TBS) and blocked with 3% bovine serum albumin in TBS. Whole lysates were precleared with agarose beads and then incubated overnight (rocking at 4°C) with cleaved Notch1 antibody. Whole cell lysates were added onto new pellets of agarose beads (previously blocked with bovine serum albumin) and incubated for 3 h in a rocking wheel at 4°C. Finally, the supernatant was removed and beads were washed several times with cold 1 × TBS-T (TBS supplemented with 0.05% Tween). Proteins were eluted with 2 × Laemmli buffer supplemented with dithiothreitol and denatured for 5 min at 99°C.
7
Molecular Signaling Pathway Analysis
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Cells were lysed in RIPA buffer protease inhibitor mixture Complete Mini (Roche) and total protein concentration was quantified by Bradford assay (Bio-Rad). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences). Samples were probed with antibodies against RET (C-20), p-RET (Tyr1062), Notch1 (C-20), HES1 (H-140), Deltex1 (D-20) from Santa Cruz, cleaved Notch1 (Val1744) (Cell Signaling), NF-κB p65 and NF-κB p65 (pS536) from BD Biosciences, TATA-binding protein (TBP; Abcam), and β-Actin (Sigma).
For immunofluorescence, cells were grown on glass slides for 24 h and fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 10% FCS and 1% BSA in PBS. Cells were incubated with primary antibody for NOTCH1, HES1, Deltex1 and NF-κB p65 (Santa Cruz) diluted in 3% BSA in PBS/0.02% Triton X-100, washed in PBS, and incubated for 60 min with fluorescence-labelled secondary antibody (Thermo Scientific). Cell nuclei were stained with DAPI solution. Images were obtained using an inverted confocal laser scanning microscope (Zeiss).
For immunofluorescence, cells were grown on glass slides for 24 h and fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 10% FCS and 1% BSA in PBS. Cells were incubated with primary antibody for NOTCH1, HES1, Deltex1 and NF-κB p65 (Santa Cruz) diluted in 3% BSA in PBS/0.02% Triton X-100, washed in PBS, and incubated for 60 min with fluorescence-labelled secondary antibody (Thermo Scientific). Cell nuclei were stained with DAPI solution. Images were obtained using an inverted confocal laser scanning microscope (Zeiss).
8
Western Blot Analysis of Protein Expression
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Total protein was extracted from approximately 2×106 cultured cells with RIPA buffer, and nuclear protein was extracted using NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL) following the manufacturer's supplied protocol. In total, 40–80 μg of the proteins was separated by electrophoresis and then transferred onto a PVDF membrane, as previously described [21] (link). The membrane was blocked with 5% defatted milk and incubated with a primary antibody against DLK1 (Proteintech Group, Chicago, IL), NOTCH1 (Cell Signaling Technology, Danvers, MA), cleaved NOTCH1 (Val1744, Cell Signaling Technology, Danvers, MA) or MMP9 (Aviva Systems Biology, San Diego, CA). The anti-Notch intracellular domain (NICD) antibody used in this study can only recognize cleaved and activated NOTCH1 but not full-length NOTCH1. After washing with PBS containing 0.1% Tween-20 (PBST), the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark), and the abundance of proteins was detected with chemiluminescence reagents. The membrane was reprobed with an antibody against GAPDH (KangChen Bio-Tech, Shanghai, China) or TBP (Abcam, Cambridge, MA) as internal controls for equivalent protein loading.
9
Profiling Molecular Markers in Tissue Microarrays
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Human tissue microarrays were purchased from Biomax, Inc. (PR8011A,PR483B). The antibodies used for IHC analysis and WB were antiactivated Notch1 (Abcam) (IHC), Cleaved Notch1 (Val 1744) (Cell Signaling Technology; 1:250 dilution), Notch1 (D1E11) (Cell Signaling Technology; 1:1,000) (WB), PTEN (Cell Signaling Technology; 1:1,000) (WB), PTEN (51–2,400; Invitrogen) (IHC), Ki-67 (Clone SP6; Lab Vision) (IHC), HSP90 (Cell Signaling Technology; 1:1,000), ADAM10 (Abcam; 1:1,000), ADAM17 (Abcam; 1:1,000) (IHC/WB), CathepsinL (Abcam; 1:1,000) (WB), CUX1 a.a. 1,300 (Millipore, 1:2,500) and CUX1 a.a. 861 (Millipore; 1:1,000) (WB), Skp2 (Santa Cruz; 1:500) (WB), p27 (Santa Cruz; 1:500) (WB), Hes1 (Santa Cruz; 1:500) (WB), c-Myc (Santa Cruz; 1:500) (WB), p21 (Santa Cruz; 1:500) (WB) and β-actin (Sigma; 1:5,000) (WB). IF analysis in prostate tissues was performed by using Vimentin (Abcam, 1:350) and E-cadherin (BD Biosciences 1:400) antibodies.
10
Detailed Western Blot Protocol
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