After 1 h of infection, the cultures were treated with GW788388 (0.5–2 μM) (Glaxo Smith Kline), recombinant TGF‐β1 (10 ng/ml) (R&D systems) or anti‐TGF‐β1 antibody (10 ng/ml) (R&D systems) and maintained for 48 h at 37°C in 5% CO2 atmosphere. Afterwards, the culture supernatants were collected and stored at −20°C for ELISA or −70°C for RT‐qPCR assays. GW788388 (4‐{4‐[3‐(pyridin‐2‐yl)−1H‐pyrazol‐4‐yl] pyridin‐2‐yl}‐N‐(tetrahydro‐2H‐pyran‐4‐yl) benzamide) (Figure 1 ) is a pharmacological selective inhibitor of TGF‐β1 types I and II receptors (TβRI/II).28
Anti tgf β1 antibody
Manufactured by R&D Systems
Sourced in United States
The Anti-TGF-β1 antibody is a laboratory reagent used to detect and quantify the transforming growth factor beta 1 (TGF-β1) protein. It is a specific antibody that binds to and identifies the TGF-β1 protein, allowing researchers to measure its levels or track its expression in various experimental systems.
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Lab products found in correlation
Recombinant tgf β1, by R&D Systems (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Anti tgf β1, by R&D Systems (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Gkt137831, by Cayman Chemical (1 mentions)
β actin, by Merck Group (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
Cremophor el, by Merck Group (1 mentions)
Recombinant tgf β1 protein, by R&D Systems (1 mentions)
Real time pcr master mix, by Roche (1 mentions)
Dihydroethidium, by Merck Group (1 mentions)
Avidin biotin peroxidase complex method, by Santa Cruz Biotechnology (1 mentions)
7 protocols using anti tgf β1 antibody
1
TGF-β1 Inhibition Modulates Cell Response
2
Anti-TGF-β1 Antibody Intervention in Mice
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1.0 mg/kg of Anti-TGF-β1 antibody (R&D Systems, Minneapolis, MN) or 1.0 mg/kg of IgG isotype antibody(R&D Systems) as control was administered intraperitoneally into Dp challenged WT and SP-D−/− mouse twice weekly from days 14 to 37 (Figure 1 ).
3
Quantification of Tube Formation and Cell Migration
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Tube formation assays were performed as described previously28 (link). Tube formation was monitored for 12–16 h under a phase-contrast microscope and quantified by counting the number of master junctions in four separate experiments in a blinded manner using ImageJ software. For Transwell migration assays, 1 × 105 HUVECs in 200 μl of serum-free M199 were seeded into the upper inserts (8-μm pores) of 12-well plates (Cat# 353182, Corning), and 600 μl of M199 supplemented with 20% FBS was added to the lower chamber. HUVECs or MCECs were then treated with 1 μg/ml PBS (control), 1 μg/ml LRG1, 5 ng/ml TGF-β1 (Cat# 240-B, R&D Systems), 10 μg/ml anti-TGF-β1 antibody (Cat# MAB1835, R&D Systems), and 5 ng/ml TGF-β1 + 10 μg/ml anti-TGF-β1 antibody (premixed overnight for neutralization). After 18 h of culture at 37 °C, non-migrated cells on the upper surface of the insert were removed with a cotton swab. Migrated cells were fixed and stained with crystal violet (Cat# V5265, Sigma) and then eluted with 10% acetic acid. The number of migrated or invaded cells was counted in four random fields.
4
Multimodal Analysis of Cellular Signaling and Oxidative Stress
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Western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF) and dihydroethidium (DHE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed as described elsewhere20 (link),23 (link),30 (link),49 (link). The antibodies used for IHC and IF included the following: anti-Hhip (monoclonal clone 5D11), α-smooth muscle actin (α-SMA) and β-actin antibodies from Sigma-Aldrich Canada; Shh, TGFβ receptor II (TGFβRII), CD31, synaptopodin (Synpo) (P-19) and p57 (H-91) antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA); p27Kip1 antibody from BD Biosciences (San Jose, CA, USA); cleaved caspase-3 (Asp175) and caspase-3 antibodies from Cell Signaling (Danvers, MA, USA); anti-TGFβ1 antibody from R&D Systems, Inc. (Burlington, ON, Canada); Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) antibody (New England Biolabs, Whitby, ON, Canada); Smad2/3 antibody (Cedarlane-Millipore, Burlington, ON, Canada); and anti-NADPH oxidase 4 (Nox4) antibody (Abcam, Cambridge, MA, USA). GKT137831 (dual inhibitor of both Nox1 and Nox4) was procured from Cayman Chemical (Ann Arbor, MI, USA).
5
Paclitaxel Formulation and Anti-TGF-β1 Antibody Preparation
6
Autonomous Synthesis and Characterization of IMB-S7
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IMB-S7 was synthesized autonomously in our laboratory, and its purity was more than 98% (HPLC). The pcDNA3.1-KLF5 plasmid was purchased from Youbio (Changsha, China), and pCMV3-Myc-ITGAV was purchased from Sino Biological Inc. (Beijing, China); pcDNA3.1-SP1 was kindly provided by Dr Li Wang (Institute of Medicinal Biotechnology, China). Real-time PCR master mix was purchased from Roche (Indianapolis, USA).
Integrin αv antibody was purchased from BD Biosciences (Becton, USA). Antibodies against Sp1, phosphor-Smad2/3, Smad2, and GAPDH were purchased from Cell Signaling Technology (Danvers, USA). Anti-α-SMA and anti-COL1A1 antibodies were obtained from Abnova (Taipei, Chinese Taiwan). Anti-TGF-β1 antibody and recombinant TGF-β1 protein were acquired from R&D Systems (Minneapolis, USA).
Integrin αv antibody was purchased from BD Biosciences (Becton, USA). Antibodies against Sp1, phosphor-Smad2/3, Smad2, and GAPDH were purchased from Cell Signaling Technology (Danvers, USA). Anti-α-SMA and anti-COL1A1 antibodies were obtained from Abnova (Taipei, Chinese Taiwan). Anti-TGF-β1 antibody and recombinant TGF-β1 protein were acquired from R&D Systems (Minneapolis, USA).
7
Quantitative Renal Morphology Analysis
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Articles displayed in microscopic fields (N = 10 fields per animal) were quantitated in a randomized and blinded fashion. Renal morphology was assessed with periodic acid-Schiff and Masson's trichrome staining (9, 10) . IHC was performed by the standard avidin-biotin-peroxidase complex method (Santa Cruz Biotechnologies, Santa Cruz, CA), as described elsewhere (9, 10) . The antibodies used included polyclonal p53 and aquaporin 1 (Santa Cruz Biotechnologies); anti-TGFβ1 antibody (R&D Systems, Burlington, Canada); monoclonal anticollagen type IV antibody (Chemicon International, Temecula, CA); polyclonal CD36 antibody (Abcam, Cambridge, MA); Fabp4 antibody (R&D Systems). Oxidative stress in vivo was assessed by dihydroethidium (Sigma-Aldrich, Oakville, Canada) staining in frozen kidney sections as reported previously (13) . The classic scoring of glomerulosclerosis (scale from 0 to 4) (39) and tubulo-interstitial injury (scale from 0 to 3) (40) was based on periodic acid-Schiff images.
The semi-quantitation of the relative staining values was performed by NIH Image J software (Bethesda, MD) (9, 10) . The images (N = 6-8 per animal) were analyzed and quantitated in a randomized and blinded fashion.
The semi-quantitation of the relative staining values was performed by NIH Image J software (Bethesda, MD) (9, 10) . The images (N = 6-8 per animal) were analyzed and quantitated in a randomized and blinded fashion.
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