The nasal mucosa slices were dehydrated and dewaxed followed by treatment with 3% hydrogen peroxide for 10 min at room temperature to quench the endogenous peroxidase activity. Then, 10% goat serum (cat. no. 5425S; Cell Signaling Technology, Inc.) and 0.3% Triton X-100 PBS solution were used to block the slices for 1 h at room temperature, followed by culturing with primary antibodies [Smad7 (cat. no. ab272928; 1:100) and TGF-β (cat. no. ab15537; 5 µg/ml); both Abcam] overnight at 4°C. Subsequently, corresponding fluorescent-labeled secondary antibody (cat. no. ab150117; 1:200; Abcam) was used to treat the slices for 1 h at room temperature. To identify the nuclei, 5 µg/ml DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) was used to stain the slices for 5 min at room temperature. Finally, a fluorescence microscope (Olympus Corporation; cat. no. BX 51; magnification, ×200) was used to observe Smad7- and TGF-β-positive cells in the nasal mucosa slices. Data were evaluated by Image ProPlus version 5.0 software (Media Cybernetics, Inc.).
Smad7
Manufactured by Cell Signaling Technology
Sourced in United States
Smad7 is a protein that plays a key role in the transforming growth factor-beta (TGF-β) signaling pathway. It functions as an inhibitor of the TGF-β signaling cascade.
Automatically generated - may contain errors
Lab products found in correlation
Smad2, by Cell Signaling Technology (4 mentions)
Smad3, by Cell Signaling Technology (4 mentions)
Smad4, by Cell Signaling Technology (3 mentions)
Smad2 3, by Cell Signaling Technology (3 mentions)
P smad2, by Cell Signaling Technology (3 mentions)
P smad3, by Cell Signaling Technology (3 mentions)
P nf κb, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Nf κb, by Cell Signaling Technology (2 mentions)
P smad2 3, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Image pro plus version 5, by Media Cybernetics (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Phospho smad2 3, by Cell Signaling Technology (1 mentions)
Primary antibodies against β actin, by Merck Group (1 mentions)
Phospho smad3, by Cell Signaling Technology (1 mentions)
12 protocols using smad7
1
Immunofluorescence Analysis of Nasal Mucosa
2
Signaling Pathway Protein Analysis
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Primary antibodies against Smad2/3, phospho-Smad2/3, Smad2, Smad3, phospho-Smad2, phospho-Smad3, Smad4, Smad7, Snail, Slug, E-cadherin, ZEB1, N-cadherin, Vimentin, NF-κB, pNFκB, IκB, phospho-IκB, IKKα, IKKβ, phospho-IKKα/β, and were purchased from Cell Signaling Technology (Danvers, MA). The primary antibodies against β-actin were purchased from Sigma Chemical Co. (St. Louis, MO). A detailed list of all antibodies used is presented in Supplementary Table S4 .
3
Western Blot Analysis of EMT Markers
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AGS cells were collected with cell lysis buffer containing protease inhibitor (Roche Applied Science, Mannheim, Germany). The cells were centrifuged for 15 minutes at 13,000 rpm. The concentration of protein was determined using a Pierce BCA Protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). The protein was loaded 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were incubated with the primary antibodies (diluted 1:1,000). Then, membranes were washed and incubated with peroxidase-conjugated secondary antibodies. Membranes were rewashed, and then were visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA). The primary antibodies used for the Western blot analysis are as follows: E-cadherin from BD Bioscience (Franklin Lakes, NJ, USA). TGF-β receptor I (TGF-βRI), N-cadherin, vimentin, snail, Twist, Zinc finger E-box binding homeobox (ZEB) 1, ZEB2 (also known as Smad interacting protein 1) , TFF1, matrix metalloproteinase (MMP)-2, MMP-7 and MMP-9 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-Smad2, p-Smad3, Smad2/3 and Smad7 from Cell Signaling Technology (Danvers, MA, USA).
4
Histological Analysis of Spinal Cord
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Spinal cord samples were fixed overnight in 4% buffered paraformaldehyde solution. After fixation, tissues were processed routinely by washing, dehydration, clearing, embedding, and freezing at the optimal cutting temperature. Slices (5 µm) were sectioned by a vibratome and stained with H&E. For immunohistochemistry, spinal cord tissue sections were deparaffinized with xylene and hydrated in ethanol solutions before heating in citrate buffer. After three extensive washes with phosphate-buffered saline (PBS), they were heated in citrate buffer for antigen retrieval, and blocked in 5% goat serum for 45 min. After rinsing in PBS, the sections were incubated overnight at 4°C with one of the following primary antibodies: chondroitin sulfate proteoglycans (CSPGs) (1:200; Abcam) or Smad7 (1:500; Cell Signaling Technology). Subsequently, sections were incubated in horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:2,500; Proteintech) for 20 min at 37°C. After three extensive washes with PBS, the sections were stained with 3,3'-diaminobenzidine tetrahydrochloride (Gene Tech, Shanghai, China), and analyzed under an Olympus microscope (Olympus Corporation, Tokyo, Japan).
5
Investigating Intestinal Stem Cell Signaling
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ISCs were seeded into Nunclon™ 35mm petri dishes and either cultured alone, or in the presence of shRNA, or rhReg1, or U0126 (10μM, Sigma, CA, USA), or LY294002 (10μM, Sigma, CA, USA), or SB431542 (10μM, Selleck, Houston, USA) for 24 hours unless otherwise specified. After 24 hours, ISCs were lysed with ice-cold lysis buffer supplemented with protease inhibitors (Roche, Basel, Switzerland). After protein content determination and separation with 12%(w/v) SDS-PAGE, Western blotting was performed, as described previously [25 (link)], using antibody against primary antibody in 2.5% non-fat dried milk in Tris-buffered saline with Tween-20 (TBST) buffer. The primary antibody were as follows: Reg1 (1:3000, Abcam, Cambridge, UK), EXTL3 (1:200, Santacruz, Dallas, USA), α-SMA (1:1000, Abcam, Cambridge, UK), Col-I (1:5000, Abcam, Cambridge, UK), Col-III (1:5000, Abcam, Cambridge, UK), FN (1:5000, Abcam, Cambridge, UK), TGF-β, Akt, P-Akt, Erk, P-Erk, Smad2/3, P-Smad2/3, Smad7 (1:1000, Cell Signaling, MA, USA), β-Actin (1:2000, Sigma, CA, USA).
6
Signaling Pathway Protein Analysis
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Antibodies to p-STAT3, STAT3, p-Smad3, Smad3, Smad7, p-PDGFR-β, PDGFR-β, p-EGFR (Tyr-845), p-NF-κB, NF-κB, p-53, fibronectin, were purchased from Cell Signaling Technology (Danvers, MA). Antibodies to Brd4, CD68, collagen 1(A2), c-Myc,MCP-1,GAPDH and the immunohistochemistry kit were purchased from Santa Cruz Biotechnology, CA. Small interfering RNA (siRNA) specific for Brd4 and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA). Antibodies to α-SMA and α-tubulin, and all other chemicals were obtained from Sigma (St. Louis, MO).
7
Western Blot Analysis of LTBP4, P-SMAD7, and SMAD7
8
Transcriptional Regulation of EMT
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Lipofectamine 3000 transfection reagent, TRIzol LS, and TGFβ1 were purchased from Invitrogen. Phospho-Smad1/5/9, Smad1, Smad2, Smad3, Smad4, Smad7, and Smad9 antibodies were from Cell Signaling Technology. E-Cadherin, α-Catenin, N-cadherin, Vimentin, Snail1, and HIF1α antibodies were from Abcam. Sox2, Oct4, CD133, and β-tubulin antibodies were from Millipore. Melatonin was purchased from Sigma. CCL20 was obtained from Peprotec. Smad1 and Smad7 plasmid constructs were purchased from Addgene.
9
Western Blot Analysis of Key Signaling Pathways
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The experimental steps of Western blot were conducted by our previous literature.21 (link) The primary antibodies listed below were incubated with the membranes at 4 °C and stayed overnight (at a 1:1,000 dilution): α-SMA, transforming growth factor (TGF)-β1, collagen-I, collagen-III, MMP-2, MMP-9, NLRP3, Caspase-1, GSDMD-N, interleukin (IL)-1β, IL-17A, and IL-18 (ABclonal Technology, Wuhan, China); ODC, SSAT, p-Smad-2, t-Smad-2, p-Smad-3, t-Smad-3, and Smad-7 (Cell Signaling Technology, Danvers, Massachusetts); and ubiquitin, β-actin, and β-tubulin (Santa Cruz Biotechnology). The membranes were incubated for 1 h at room temperature with secondary antibodies (diluted at 1:10,000, Proteintech, Wuhan, China). The specific complexes treated with an ECL kit (MultiSciences, Hangzhou, China) were detected using a multiplex fluorescent imaging system.
10
Western Blot Analysis of Smad Signaling
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Cell lysates were prepared in RIPA (Biyuntian, Haimen, China) lysis buffer with 1% cocktail (Biyuntian, Haimen, China). Protein concentrations were measured via the BCA method. Then cell lysate was added to each well and separated by SDS-polyacrylamide gel electrophoresis. Subsequently, the proteins were electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk for 2 h, the membranes were incubated with specific primary antibodies including anti-p-Smad3, Smad3, p-Smad2, Smad2, Smad7 (Cell Signaling Technology, USA), and β-actin (Santa Cruz, USA) at 4 °C overnight. Afterward, the membranes were incubated with HRP-conjugated secondary antibody (LICOR, USA) for 60 min. Finally, the blots were scanned with a two-color infrared Li-Cor scanner (Odyssey, LICOR, USA).
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