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Tgf βrii

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TGF-βRII is a cell surface receptor that binds to the transforming growth factor-beta (TGF-beta) ligand. It is a key component of the TGF-beta signaling pathway, which regulates various cellular processes such as cell proliferation, differentiation, and apoptosis.

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Lab products found in correlation

Tgf βri, by Abcam (3 mentions) Tgf β1, by Abcam (3 mentions) P smad2 3, by Abcam (2 mentions) Smad2 3, by Cell Signaling Technology (2 mentions) Anti klotho, by Abcam (1 mentions) Smad2 3, by Santa Cruz Biotechnology (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Collagen 4, by Abcam (1 mentions) Collagen 1, by Abcam (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) High binding microtiter plates, by Corning (1 mentions) Tgf β1, by ACROBiosystems (1 mentions) Streptavidin hrp, by Abcam (1 mentions) Microplate reader, by Molecular Devices (1 mentions) Spectramax m5 microplate reader, by Molecular Devices (1 mentions) Curcumol, by Merck Group (1 mentions) Fibronectin, by Abcam (1 mentions) Anti mouse igg, by Proteintech (1 mentions) 5 5 6 6 tetrachloro 1 1 3 3 tetraethyl benzimidazolyl carbocyanineiodide jc 1, by Beyotime (1 mentions) Timp 2, by Cell Signaling Technology (1 mentions)

7 protocols using tgf βrii

1

Western Blot Analysis of Kidney Proteins

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Total proteins were extracted from the kidneys or HK‐2 cells and the concentration was measured by BCA protein assay kit (Beyotime, Shanghai, China). Western blot assay was performed with the standard method. Briefly, proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). PVDF membrane (Millipore, Bedford, MA, USA) was used to electro‐transfer. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with primary antibodies: anti‐Klotho (1:500, Abcam, #203576), TGF‐β1 (1:1000, Abcam, #92486), TGFβ‐RI (1:800, Abcam, #31013), TGFβ‐RII (1:1000, Abcam #186838), α‐SMA (1:1000, CST, #56856), Collagen I (1:2000, Abcam, #34710), Collagen IV (1:1500, Abcam, #6586), Smad 2/3 (1:1000, Santa Cruz, #398844), p‐Smad 2/3 (1:800, Abcam, #63399), Smad 4 (1:1000, CST, #38454) and Smad 7 (1:1000, Santa Cruz, #365846), followed by incubation with secondary antibody at room temperature for 1 hour. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was used as the internal control. Protein bands were visualized by enhanced chemiluminescence (ECL) reagent and exposed using BioImaging Systems (UVP, Upland, CA, USA). The relative protein levels were quantified using the Image J software (National Institutes of Health, Montgomery, MD, USA). All the assays were performed in triplicate.
Liu M., Liu T., Shang P., Zhang Y., Liu L., Liu T, & Sun S. (2018). Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway. Journal of Cellular and Molecular Medicine, 22(10), 4997-5007.
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2

Blocking Assays for PD-1, CD80, and TGF-β Interactions

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The blocking ability of HS636 and BR102 was tested in competition ELISA assays. Fc-tagged human PD-1 (ACROBiosystems) was absorbed into high-binding microtiter plates (Corning) at 0.25 µg/mL in PBS at 4 °C overnight. The plates were blocked with 5% BSA in PBST. Biotinylated PD-L1 (ACROBiosystems) and serially diluted HS636 or BR102 were added to the plates. The samples were then incubated with Streptavidin-HRP (Abcam). TMB color development was stopped with 1M H2SO4, and the absorbance was detected at 450 nm using a Spectramax M5 microplate reader (Molecular Devices). For CD80/PD-L1 blocking assay, Fc-tagged human CD80 (ACROBiosystems) was coated at 0.5 µg/mL, and biotinylated PD-L1 was added at 4 µg/mL.
For the TGF-β1/TGF-βRII blocking assay, varying amounts of BR102 and biotinylated TGF-βRII (ACROBiosystems) were added to 96-well plates pre-coated with TGF-β1 (ACROBiosystems). Bound TGF-βRII was detected by adding Streptavidin-HRP (Abcam) followed by TMB. Absorbance at 450 nm was read on a microplate reader (Molecular Devices).
Wu Z.H., Li N., Gao Z.Z., Chen G., Nie L., Zhou Y.Q., Jiang M.Z., Chen Y., Chen J., Mei X.F., Hu F, & Wang H.B. (2022). Development of the Novel Bifunctional Fusion Protein BR102 That Simultaneously Targets PD-L1 and TGF-β for Anticancer Immunotherapy. Cancers, 14(19), 4964.
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3

Investigating Curcumol-Mediated Cellular Responses

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Curcumol, N-acetyl cysteine (NAC), N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), necrotatin-1 (Nec-1), and SP600125 were brought from Sigma-Aldrich (St Louis, MO, USA). Dulbecco's modified essential medium (DMEM), fetal bovine serum (FBS), Opti MEM medium, phosphate buffered saline (PBS), and trypsin-EDTA were purchased from GIBCO BRL (Grand Island, NY, USA). Oxidation sensitive 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were obtained by Beyotime Biotechnology (Shanghai, China). MitoSox Red was brought from Thermo Fisher Scientific (Waltham, MA, USA). Anti-rabbit IgG, anti-mouse IgG and antibodies against β-actin, and RIPK1 (17519-1-AP) were purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Main antibodies against α-SMA, α1(I) procollagen, EGFR, fibronectin, MLKL, PDGF-βR, p-MLKL, p-RIPK3, RIPK3, and TGF-βRII were obtained from Abcam Technology (Abcam, Cambridge, UK). Primary antibodies against caspase3, cleaved-caspase3, caspase8, cleaved-caspase8, caspase9, cleaved-caspase9, ERK, JNK, p38, p-ERK, p-JNK, p-p38, p-RIPK1 and TIMP2 were procured by Cell Signaling Technology (Danvers, MA, USA). Lentivirus vectors encoding negative control shRNA (NC shRNA) and RIPK3 shRNA were designed by Hanbio (Shanghai, China).
Jia Y., Wang F., Guo Q., Li M., Wang L., Zhang Z., Jiang S., Jin H., Chen A., Tan S., Zhang F., Shao J, & Zheng S. (2018). Curcumol induces RIPK1/RIPK3 complex-dependent necroptosis via JNK1/2-ROS signaling in hepatic stellate cells. Redox Biology, 19, 375-387.
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4

Evaluating TGF-β Signaling in Gata1low Mice

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BM and spleen from Gata1low mice treated for 5 days were dissolved in lysis buffer containing protease and phosphatase inhibitors and stored at −80°C. Protein extracts were separated by electrophoresis under denaturing conditions using 7.5–10% mini-Protean TGX pre-casted gels (Bio-Rad, CA, USA) and transferred to nitrocellulose filters with the Transblot-Turbo system (Bio-Rad, Hercules). Filters were probed with antibodies against proteins of the canonical (SMAD2/3, cat no. 8685, Cell Signaling, Boston, MA, USA), p-SMAD2/3 (cat no. 8828, Cell Signaling), TGF-βRII (cat no. ab186838, Abcam, Cambridge, UK) and non-canonical (p38, cat no. 9212; p-p38, cat no. 4511; ERK1/2, cat no. 9102; p-ERK1/2, cat no. 9101; all from Cell Signaling) TGF-β signaling and of the JAK/STAT signaling (JAK2 (cat. No 3230, Cell Signaling), STAT5 (cat no. sc-74442, Santa Cruz, Dallas, Texas, USA), pJAK2 (Phospho-Tyr1007/1008 JAK2, cat no. 3771, Cell Signaling) and p-STAT5 (cat no, 9351, Cell Signaling). GAPDH (cat no. G9545, Sigma Aldrich) was used as a loading control. The bands were quantified with the ImageJ 1.52q software (National Institutes of Health, Bethesda, MD, USA) and normalized against GAPDH. Stoichiometry determinations of phospho-proteins levels were obtained by normalizing the content of the phosphoprotein with that of the corresponding total protein.
Hardy L.W, & Nalivaika E. (1992). Asn177 in Escherichia coli thymidylate synthase is a major determinant of pyrimidine specificity.. Proceedings of the National Academy of Sciences of the United States of America, 89(20), 9725-9729.
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5

Exosome Isolation and Analysis

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Antibodies against rat Nrf2, TGF-β1, TGF-βRII, TGF-βRI, β-actin, TSG101, CD63, Alix, GM130, and ELISA kit for TNF-α were obtained from Abcam PLC (MA, USA). DMEM, F12 nutrient medium, fetal bovine serum (FBS) without exosome, Trizol-LS, exosomal protein extraction kit, and Annexin V-FITC Apoptosis Kit were purchased from Invitrogen (Carlsbad, USA). qRT-PCR primers, let-7d-5p mimics, miR-NC, and let-7d-5p inhibitor were from Sangon Biotech (Shanghai, China). DCFH-DA and PKH67 green fluorescent cell linker mini-kit were obtained from Sigma Chemical Company (St. Louis, USA). The kit for bicinchoninic acid (BCA) protein assay was purchased from Beyotime Biotechnology (Shanghai, China).
Gao Y., Sun J., Dong C., Zhao M., Hu Y, & Jin F. (2020). Extracellular Vesicles Derived from Adipose Mesenchymal Stem Cells Alleviate PM2.5-Induced Lung Injury and Pulmonary Fibrosis. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 26, e922782-1-e922782-13.
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6

Western Blot Analysis of Cellular Signaling

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After obtaining cell lysates from cultured cells, proteins were separated by SDS-PAGE and electroblotted onto nitrocellulose membranes. Antibodies used include phosphor-TGFβ receptor II (pTGFβRII, Thermo Scientific), CYR61, TGFβRII, p21, and p16 (all from Abcam), pRB, RB, TGFβ, pSMAD2, pSMAD3, SMAD2/3, p53, pp38, p38, phosphor-endothelial nitric oxide synthase (peNOS), eNOS, pAKT, AKT, phosphor-AMP-activated protein kinase (pAMPK), and AMPK (all from Cell Signaling). β-actin was used as a loading control (Sigma Aldrich). Band quantification was performed using ImageJ (16 (link)).
Tsou P.S., Khanna D, & Sawalha A.H. (2019). Identifying CYR61 as a Potential Anti-fibrotic and Pro-angiogenic Mediator in Scleroderma. Arthritis & rheumatology (Hoboken, N.J.), 71(8), 1350-1359.
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7

Celastrol Inhibits TGF-β1 Signaling

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Celastrol was obtained from Sigma-Aldrich (St Louis, MO, USA). The primary antibodies of TGF-β1, Smad2/3, p-Smad2/3, TGFβRI, TGFβRII, and Smad4 were purchased from Abcam (Cambridge, UK). Cell counting kit-8 (CCK-8) was purchased from Biosharp (Hefei, China). Antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and other reagents were purchased from Sigma-Aldrich.
Jiang Z., Cao Q., Dai G., Wang J., Liu C., Lv L, & Pan J. (2019). Celastrol inhibits colorectal cancer through TGF-β1/Smad signaling. OncoTargets and therapy, 12, 509-518.
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