All human CRC cell lines; CL34, Caco2, Colo-320, DLD1, HCT15, HCT116, HT29 and LOVO were obtained from American Type Culture Collection and cultured in Roswell Park Memorial Institute 1640 media supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO2. All treatment experiments were performed in reduced FBS condition (5%). Apoptosis analysis was performed using annexin V/propidium iodide dual staining and measured by flow cytometry as previously described.30 (link) Antibodies against MED12 (CST#14360), E-cadherin (CST#3195), N-cadherin (CST#14215), pERK1/2 (CST#4370), ERK1/2 (CST#4695) and β-actin (CST#3700) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Antibodies against transforming growth factor (TGF)-β-R2 (sc#17799), vimentin (sc#5565) and GAPDH (sc#25778) were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). Twist antibody (ab#175430) was purchased from Abcam (Cambridge, Massachusetts, USA).
Tgfβr2
Manufactured by Santa Cruz Biotechnology
Sourced in United States
TGFβR2 is a receptor protein that binds to the cytokine transforming growth factor beta (TGF-β). It is a crucial component of the TGF-β signaling pathway, which regulates various cellular processes such as cell growth, differentiation, and apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Cell Signaling Technology (3 mentions)
Tgfβr1, by Santa Cruz Biotechnology (3 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
Tgf β1, by Santa Cruz Biotechnology (2 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Twist antibody, by Abcam (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Dld 1, by American Type Culture Collection (1 mentions)
Caco 2, by American Type Culture Collection (1 mentions)
Hct15, by American Type Culture Collection (1 mentions)
Colo320, by American Type Culture Collection (1 mentions)
Antibodies for fibronectin, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Pfra1, by Cell Signaling Technology (1 mentions)
Vectastain abc dab kit, by Vector Laboratories (1 mentions)
P smad2, by Cell Signaling Technology (1 mentions)
7 protocols using tgfβr2
1
CRC Cell Line Culture and Apoptosis Assay
2
Protein Expression Analysis in Eyecup Cells
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The eyecups were lysed in radioimmunoprecipitation assay buffer. An equal amount of total proteins was subjected to electrophoresis on 10% SDS-PAGE and then electrophoretically transferred to a polyvinylidene fluoride film. The member was blocked with 10% non-fat milk for 2 h at room temperature (RT) and incubated with primary antibody overnight at 4°C. After several washes, the membrane was then incubated with secondary antibody for 1 h at RT. The signal was developed with an enhanced chemiluminescence reagent kit (NCM Biotech, Newport, RI, United States). The bands were quantified with an ImageJ density analyzer and normalized to GAPDH levels. Antibodies for vimentin, collagen-1, alpha-smooth muscle actin (α-SMA), and CTGF were purchased from Abcam (Cambridge, United Kingdom). Antibodies for fibronectin, Smad2/3, and TGF-β receptor 2 (TGF-βR2) were obtained from Santa Cruz Biotechnology (Dallas, TX, United States). Antibodies for glycogen synthase kinase 3 beta (GSK3β), phosphorylated-GSK3β (p-GSK3β), phosphorylated-Smad2/3 (p-Smad2/3), anti-non–phosphorylated β-catenin (non–p-β-catenin), and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, United States).
3
Generation and Characterization of Genetically Engineered Mouse Models
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The generation of p18−/−, p18+/−, and p18−/−:Brca1+/− mice in Balb/c background, and p18−/−and p18−/−;Brca1f/f; MMTV-Cre (p18−/−; Brca1MGKO) mice in Balb/c-B6 mixed background has been previously described [18 (link), 19 (link), 54 (link)]. The Institutional Animal Care and Use Committee at the University of Miami and Shenzhen University approved all animal procedures. Animals were housed in a specific pathogen-free environment with a 12/12 light cycle. Animals were euthanized by exposure to isoflurane followed by cervical dislocation. At least four female mice were analyzed for each genotype, or were transplanted with each type of tumor cells. Histopathology and immunohistochemistry (IHC) were performed as previously described [18 (link), 19 (link), 54 (link)]. The primary antibodies used were: TGFβR1, TGFβR2 (Santa Cruz), p-Smad2, p-FRA1 (Cell signaling), CK5 (Covance), and Vim (Abcam). Immunocomplexes were detected using the Vectastain ABC DAB kit according to the manufacturer’s instructions (Vector Laboratories). The positive results of IHC were quantified by H-score, as previously described [55 (link)].
4
Molecular Mechanisms of NXT-Mediated Signaling
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NXT was kindly provided by Xianyang Buchang Pharmaceutical Co. Ltd (Shan’xi, China). Rabbit anti-VEGFA, PCK1, GCK, INSR, PI3K (p110), IRS1 and pi-IRS1 polyclonal antibodies were purchased from Proteintech Group (Chicago, IL). Goat anti-AGE polyclonal antibody was purchased from Novus Biologicals (Littleton, CO). Rabbit anti-AKT, pi-AKT, pi-IRS1, IRS2, PI3K (P85), AMPKα and pi-AMPKα polyclonal antibodies were purchased from Cell Signaling Technology Inc (Danvers, MA). The following antibodies were purchased from Santa Cruz Inc.: rabbit anti-MMP2, fibronectin, G6Pase, FGF21, WT1, TGFβ1, TGFβR2 and Smad2/3 polyclonal antibodies; goat anti-MMP9, collagen type I α2 (COL1A2), collagen type IV α1/3 (COL4A1/3), pi-Smad2/3 and CTGF polyclonal antibodies; and mouse anti-GLUT4 monoclonal antibody. The mouse insulin ELISA assay kit was purchased from ABclonal Inc. (Wuhan, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) except as indicated.
5
Stem Cell Tumor Microenvironment Analysis
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Matrigel matrix (356234) was purchased from Corning. Quantikine ELISA kits for IL-6 (D6050) and IL-8 (D8000C), recombinant VEGF-A165 (293-VE-010; used at 19 ng/ml). and PE-conjugated VEGFR2 antibody for surface staining (FAB357P; IF-FACS 1:120) were purchased from R&D Systems. Antibodies to CD44 (5640; IF and IF-FACS 1:100), E-cadherin (3195; IF-FACS 1:100; WB 1:1000), Nanog (3580; WB 1:1000), Oct4 (4286; WB 1:1000), Sox-2 (2748; WB 1:1000), SCF/Kit ligand (2093; IF-FACS 1:100), PD-L1 (13684; IF-FACS 1:100), RalA (4799; WB 1:1000), and RalBP1 (5739; WB 1:1000) were purchased from Cell Signaling Technology. Antibodies to phospho-Smad2/3 (Sc-11769R; IF 1:100), TGF-βR2 (Sc-220; IF-FACS 1:100), caspase-3 (Sc-7148; WB 1:500), ABCB1/Mdr1/P-glycoprotein (Sc-8313; IF-FACS 1:100), CD24 (Sc-19585; IF-FACS 1:100), VEGFR2 (Sc-504; WB 1:500), and PKC-ζ (Sc-17781; WB 1:500) were purchased from Santa Cruz Biotechnology. Verapamil (V4629; used at 100 μM) was purchased from Sigma. Dasatinib (D-3307; used at 500 nM) was purchased from LC Laboratories.
6
Protein Expression Analysis in Liver Tissue
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The liver tissue was lysed in 0.5 mL of CelLytic M lysis reagent (Sigma-Aldrich, St. Louis, MO, USA) with 1% phosphatase inhibitor cocktail and protease inhibitor cocktail and centrifuged at 13,000× g for 30 min at 4 °C. The concentration of protein lysate was determined using the Bradford assay. In addition, the nuclear fraction was harvested using a nuclear extraction kit (Abcam, Cambridge, MA, USA) in accordance with the manufacturer’s instructions. The cell lysates were separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes, following incubation with primary antibodies against NF-κB, α-SMA, TGFβ-R1, and TGFβ-R2 (all from Santa Cruz Biotechnology, Dallas, TX, USA). Finally, horseradish peroxidase-conjugated secondary antibodies were added, and the reaction was detected by electrochemiluminescence. The data were calibrated using H1 and β-actin as internal controls.
7
Regulation of Endometrial Cancer Markers
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Endometrial cancer cells AN3CA, Nestin knockdown AN3CA, KLE, Nestin knockdown KLE, Ishikawa, and Nestin overexpressing Ishikawa cells extracts were analyzed using antibodies against Nestin, cyclin D1, cyclin D3, p27, p21 (Cell Signaling Technology), TGFβ1, TGFβ2, TGFβ3, TGFβR1, TGFβR2, TGFβR3, pSMAD2/3, SMAD2/3, SMAD4 (Santa Cruz Biotechnology), pFAK (phospho Y397), E-cadherin, N-cadherin, SNAIL, SLUG, Twist, vimentin (Cell Signaling Technology, Inc.), progesterone receptor (PgR 1294; Dako Corporation), and β-actin (Sigma-Aldrich). Equal amounts of protein were subjected to SDS-PAGE. The enhanced chemiluminescence system was used to visualize the protein bands as recommended by the manufacturer (Pierce). Protein bands were quantified using densitometry software (Bio-Rad) and normalized by using actin as a loading control. To calculate the relative intensity of each band individual bands were divided by the corresponding loading control intensity.
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