Dendrobium officinale was purchased from Zhejiang Shouxiangu Pharmaceutical Co., Ltd. (Jinhua, China), and PMA and LPS were purchased from Sigma (Saint Louis, MI, USA). Recombinant human interleukin-4 (IL-4) was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). TNF-α and IL-6 ELISA kits were purchased from Lianke Biological Technology (Hangzhou, China). Cell Counting Kit-8 (CCK-8) Assay Kit was received from MCE (Romulus, NJ, USA). Total Protein Extraction kit was purchased from Beyotime (Shanghai, China). The MiNiBEST Universal RNA Extraction Kit, PrimeScript RT Reagent kit, and SYBR Premix Ex Taq II Kit were purchased from TaKaRa (Dalian, China). Transwell polycarbonate membrane had an 8 μm pore size (Corning City, NY, USA). The primary antibodies E-cadherin, N-cadherin, Vimentin, Caspase-3, Bax, Bcl-2, Ki67, and β-actin were purchased from Proteintech (Wuhan, China). The primary antibodies ARG1, TGM2, and Cleaved-NOTCH1 were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies STAT6, p-STAT6, PPAR-r, JAGGED1, Cleaved-NOTCH1, and NOTCH1 were purchased from Abcam (Cambridge, UK). β-actin was purchased from Proteintech (Wuhan, China). The ECL Plus Western Blotting Detection Kit was purchased from Technology Co., Ltd. (Beijing, China). Anti-CD80-FITC and Anti-CD206-PE were obtained from Thermo Fisher (Waltham, MA, USA).
Cleaved notch1
Manufactured by Abcam
Sourced in United Kingdom
Cleaved Notch1 is a recombinant protein fragment that represents the cleaved intracellular domain of the Notch1 receptor. It is produced in E. coli and purified using standard protocols.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved notch1, by Cell Signaling Technology (2 mentions)
Notch1, by Abcam (2 mentions)
Pthr735 adam17, by Merck Group (2 mentions)
Total protein extraction kit, by Beyotime (1 mentions)
Stat6, by Abcam (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Minibest universal rna extraction kit, by Takara Bio (1 mentions)
P stat6, by Abcam (1 mentions)
Recombinant human interleukin 4 il 4, by Thermo Fisher Scientific (1 mentions)
Ppar r, by Abcam (1 mentions)
Jagged1, by Abcam (1 mentions)
Anti cd206 pe, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Caspase 3, by Proteintech (1 mentions)
8 protocols using cleaved notch1
1
Dendrobium officinale Immune Regulation Protocol
2
Western Blot Analysis of Notch Signaling
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Western blot analysis was performed as described previously [26 (link)]. Primary antibodies include Notch1 (CST, cat#3608), cleaved Notch1 (CST, Val1744, D3B8, cat#4147), Hes1 (CST, cat#11988), Hey1 (Abcam, ab154077), Nanog (CST, cat#4903), Sox2 (CST, cat#3579), Oct4 (CST, cat#2750), MSI2 (Abcam, ab76148), LFNG (CST, cat #66472), Numb (CST, cat #2756). Anti-β-actin (Abcam, ab8226) was used as an internal control. Immune complexes were visualized using the Beyo ECL Plus.
3
Immunohistochemical Analysis of Kidney Tissues
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Kidney tissues were fixed in 4% PBS buffered formaldehyde, embedded in paraffin, and then sliced longitudinally into 4-μm thick sections. Antibodies against phosphorylated JNK (R & D Systems, Minneapolis, MN), cleaved Notch-1, cleaved Notch-2, α-SMA and fibronectin (Abcam, Cambridge, UK) were used for immunohistochemistry. Immunoreactivity in sections was detected using a horseradish peroxidase-3′-,3′-diaminobenzidine kit (SuperPicture™ Kit, Invitrogen, Carlsbad, CA).
4
Protein Expression Analysis of Notch Signaling
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The concentration of extracted protein was detected by Protein BCA Assay (Beyotime, Jiangsu, China). Protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After blocking with nonfat milk, primary antibodies specific for Jagged-1 (Cell Signaling Technology, CST, Boston, USA), full-length Notch-1 (CST), cleaved Notch-1 (NICD, Abcam, Cambridge, UK), Synaptopodin (Abcam), Hes-1 (Abcam), VEGF (Abcam), p53 (CST), p62 (Abcam), LC3B (Abcam) were used. Goat anti-rabbit IgG conjugated with horseradish peroxidase was used to detect the expression of primary antibodies, and protein bands were visualized by ECL western blotting detection system (Amersham, Little Chalfont, UK).
5
Histological and IHC Analyses of Tissues
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For histological analyses, tissues were fixed in 10% buffered formalin and embedded in paraffin. Hematoxilin and eosin (H&E) staining and IHC analyses were performed on 3-µm paraffin sections. For IHC, the following antibodies were used: CD3 (Pharmingen, 553057), cleaved Notch1 (Abcam, ab8925), Hes1 (CNIO), TDT (DAKO, A3524), Ki67 (Master Diagnostica, 0003110QD), γH2AX (Millipore, 05-636), and active Caspase 3 (Cell Signaling, 9661). PAS staining was performed by incubation for 5 min with periodic acid followed by 20 min of incubation with Schiff reagent and hematoxylin counterstaining.
6
Quantitative Protein Analysis of Lung Tissues
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Total protein lysates were prepared from snap‐frozen lung tissues or cell lysates and subjected to ELISA and immunoblotting. Human and mouse IL‐6R ELISA sets were purchased from R&D Systems. Immunoblotting was performed with the following antibodies: total ADAM17 (from S. Rose‐John), pThr735‐ADAM17 (Sigma), Myc, pTyr705‐STAT3, pSer727‐STAT3, total STAT3, pSer473‐AKT, total AKT, pThr202/pTyr204‐ERK1/2, total ERK1/2, pThr180/pTyr182‐p38 MAPK, total p38 MAPK, pTyr1068‐EGFR, total EGFR, pSer82‐HSP27 (Cell Signaling Technology), cleaved Notch1 (Abcam), IL‐6R, Nrg1, pTyr1289‐ErbB3, total ErbB3, TGFα (Santa Cruz Biotechnology), and actin (Sigma). Protein bands were visualized using the Odyssey Infrared Imaging System (LI‐COR) and quantified using Image J. Antibody dilutions are indicated in Appendix Table S2 .
7
Immunostaining for ADAM17 and MAPK Pathway
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Formalin‐fixed, paraffin‐embedded (FFPE) human lung sections were stained with antibodies against pThr735‐ADAM17 (Sigma), as well as pThr202/pTyr204‐ERK1/2 and pThr180/pTyr182‐p38 MAPK (Cell Signaling Technology). FFPE mouse lung sections were subjected to histologic evaluation by staining with hematoxylin and eosin (H&E), as well as IHC with the following antibodies: B220, CD3, CD45, and Ly6G (BD Biosciences); CD31 and TTF‐1 (Abcam); PCNA, cleaved Caspase‐3, and pThr202/pTyr204‐ERK1/2 (Cell Signaling Technology); pThr735‐ADAM17 (Sigma); and F4/80 (Bio‐Rad). To quantify cellular staining, digital images of photomicrographs (60× high‐power fields) were viewed using Image J software. Positive‐staining cells were counted manually (n = 20 fields). FFPE xenograft sections were subjected to IHC evaluation by staining with pThr735‐ADAM17 (Sigma), pThr202/pTyr204‐ERK1/2 (Cell Signaling Technology), and cleaved Notch1 (Abcam) antibodies. Antibody dilutions are indicated in Appendix Table S2 .
8
Cytoplasmic, Nuclear, and Chromatin Fractionation
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Cells were lysed in 20min at 4C in PBS plus 0.5% Triton X-100, 1mM EDTA, 100 mM Naorthovanadate and protease inhibitors (Roche). For cytoplasm/nuclear/chromatin separations, 10mM HEPES, 1.5mM MgCl2, 10mM KCl, 0.05%NP40 at pH 7.9, 10min on ice and centrifuged at 13.000 rpm. Supernatants were recovered and pellets lysed in 5mM HEPES, 1.5mM MgCl2, 0.2 mM EDTA, 0.5mM DTT and 26% glycerol and sonicated three times. Lysates were analyzed by western blot. β-Catenin (c2206, Sigma), cleaved Notch1 (#2421, Cell Signaling) and Kaiso (ab12723, Abcam) antibodies were used.
For immunoprecipitations, cells were lysed 30min at 4ºC in 300μl PBS plus 0.5% Triton X-100, 1mM EDTA, 100 mM Na-orthovanadate, 0.25 mM PMSF and complete protease inhibitor cocktail (Roche). After centrifugation, supernatants were pre-cleared 2h with 1% of BSA, 1μg
IgGs and 50μL SPA beads. Precleared lysates were incubated O/N with 3μg of indicated antibodies (Irrelevant IgG or anti-β-Catenin (BD Bioscience, catalog no. 610154) or cleaved Notch1 (ab8925, Abcam)). Antibody-Protein complexes were captured with 30μL SPA beads for 2h. After washing, precipitates were analyzed by western blot.
For immunoprecipitations, cells were lysed 30min at 4ºC in 300μl PBS plus 0.5% Triton X-100, 1mM EDTA, 100 mM Na-orthovanadate, 0.25 mM PMSF and complete protease inhibitor cocktail (Roche). After centrifugation, supernatants were pre-cleared 2h with 1% of BSA, 1μg
IgGs and 50μL SPA beads. Precleared lysates were incubated O/N with 3μg of indicated antibodies (Irrelevant IgG or anti-β-Catenin (BD Bioscience, catalog no. 610154) or cleaved Notch1 (ab8925, Abcam)). Antibody-Protein complexes were captured with 30μL SPA beads for 2h. After washing, precipitates were analyzed by western blot.
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