Rabbit polyclonal anti‐α‐SMA was acquired from Abcam. Rabbit polyclonal anti‐GAPDH, anti‐von Willebrand Factor (vWF), and anti‐fibroblast‐specific protein 1 (FSP1) were acquired from Santa Cruz Biotechnology. Rabbit Polyclonal Endothelin‐1 was purchased from Fisher Scientific. Rabbit polyclonal anti‐BMPRII and anti‐P‐SMAD1/5/8 were purchased from Cell Signaling Technology. Mouse monoclonal anti‐eNOS, anti‐β‐actin and rabbit polyclonal anti‐Cav‐1 were purchased from BD PharMingen. Alexa‐Fluor 488 and 555‐conjugated goat and donkey antimouse or antirabbit IgG were purchased from Life Technologies. Antimouse and antirabbit HRP‐conjugated IgG were purchased from Cell Signaling Technology or Kierkegaard and Perry Laboratories (Supporting Information 1 ). RIPA buffer, protease, and phosphatase inhibitor cocktail, collagenase type I, paraformaldehyde, heparin, and sucrose were purchased from SIGMA Chemical, Co. Mounting media containing DAPI (VectaShield) was obtained from Vector. Stock solutions were prepared in 100% dimethylsulfoxide (DMSO), buffered physiological solution or sterile phosphate‐buffered saline (PBS) and diluted daily in sterile PBS or DMEM. The highest final concentration of the solvent was 0.1% (v/v) and did not affect the experiments. PCR primers were purchased from Integrated DNA Technologies, Inc.
Anti p smad1 5 8
Manufactured by Cell Signaling Technology
Sourced in United States, Germany
Anti-p-Smad1/5/8 is a lab equipment product that detects phosphorylated (active) forms of Smad1, Smad5, and Smad8 proteins. Smad proteins are key mediators of the transforming growth factor-beta (TGF-β) signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti p smad2 3, by Cell Signaling Technology (4 mentions)
Anti smad1, by Cell Signaling Technology (3 mentions)
Anti p stat3, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti smad4, by Cell Signaling Technology (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Anti runx2, by Cell Signaling Technology (2 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Anti β catenin, by Merck Group (2 mentions)
Anti p65, by Cell Signaling Technology (2 mentions)
Anti gsk3β, by Abcam (2 mentions)
Anti axin1, by Cell Signaling Technology (2 mentions)
Anti dkk1, by Santa Cruz Biotechnology (2 mentions)
Anti stat3, by Cell Signaling Technology (2 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Sucrose, by Merck Group (1 mentions)
20 protocols using anti p smad1 5 8
1
Antibody and Reagent Sources for Cell Analysis
2
Immunofluorescence Staining of Smad1/5/8 and β-catenin
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Cells were grown on glass coverslips and incubated with TMDQ for 48 h. Cells were fixed in 10% formalin for 15 min at room temperature. After washing three times in 1 × PBS, the cells were permeabilized with 0.2% Triton X-100 in 1 × PBS for 20 min, washed three times in 1 × PBS, and then blocked with 5% BSA in 1 × PBS for 1 h at room temperature. After then, the cells were incubated with anti-p-Smad1/5/8 (1 : 200, Cell Signaling) and anti- β-catenin (1 : 200, Abcam) antibodies for overnight at room temperature, washed three times, and incubated with Alexa-488-conjugated secondary antibodies (1 : 500, Invitrogen) for 2 h at room temperature. The cells was stained with DAPI (Sigma-Aldrich) and washed three times, mounted on glass slides, and viewed on confocal microscopy (Cell Voyager, Yokohama, Japan).
3
Immunohistochemical and Western Blot Analyses
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Anti-p-PDHE1α (S293, S300) (Calbiochem, San Diego, CA, USA, AP1062-1064, respectively) were used for immunohistochemistry. Anti-p-PDHE1α (S293, S300) (Abfrontier), Anti-SMAD1/5/8 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-6031-R), anti-p-SMAD 1/5/8 (Cell Signaling Technology, Danvers, MA, USA, #9511), COXIV (Abcam, Cambridge, UK, ab16056) and anti-Flag (Sigma-Aldrich, F1804) antibodies were used for both immunofluorescence and western blotting. Anti-SMAD1 (Invitrogen, Carlsbad, CA, USA, #38-5400), anti-SMAD5 (Abgent, San Diego, CA, USA, AJ1726a), and Anti-SMAD1/5 (Santa Cruz Biotechnology, sc-6201) were used for immunofluorescence. Anti-α-tubulin (Applied Biological Materials, G098), anti-lamin B (Santa Cruz Biotechnology, sc-6216), anti-SMAD4 (Cell Signaling Technology, #9515) and anti-PDK4 serum (Abfrontier) were used for western blotting.
4
Immunohistochemical Staining of Shh and pSmad
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Fixed tissues were sectioned and incubated with primary antibodies: anti-Shh 5E1 (1:50; Developmental Studies Hybridoma Bank); anti-pSmad 1/5/8 (1:100-1:500; Cell Signaling Technologies, cat. no. 9511). Alexa 488 and 594 conjugated secondary antibodies were used (1:500; ThermoFisher Scientific/Molecular Probes, cat. nos. A11001, A11034 and A11005). Slides were mounted in Vectashield (Vector Laboratories) and analysed.
5
Immunofluorescence and X-gal staining
6
Immunohistochemical Analysis of Embryonic Development
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Embryos and explants were analysed by immunohistochemistry according to standard techniques (Manning et al., 2006 (link)). Embryos or cryosectioned sections were analysed using the following antibodies anti-pSMAD1/5/8 (1:500, Cell Signaling Technology, 9511), anti-FOXA2 (1:50, DSHB, 2C7), anti-LHX3 (1:50, DSHB, 64.4E12/67.4E12), anti-SHH (1:50, DSHB, 5E1), anti-ISLET1 (1:50, DSHB, 4D5.65) and anti-PH3 (1:1000, Cell Signaling Technology, 06-570). Secondary antibodies (1:500, Jackson ImmunoResearch) were conjugated with anti-Alexa 488 or 594. Images were taken using a Zeiss Apotome.
7
Whole Mount Immunohistochemistry Protocol
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Immunohistochemistry was performed according to a standard whole mount immunohistochemistry protocol. Briefly, embryos/hatchlings were fixed, washed, bleached (KOH/H2O2 in PTW), and blocked (BSA [1%], DMSO [1%], Triton X-100 [0.1%], NGS [4%], PBS [1×]). In case of anti-pSmad 1/5/8 immunohistochemistry embryos were additionally treated with proteinase K (10 µg/ml, 16.5 hpf: 5 min, 19 hpf and 21.5 hpf: 6 min). Samples were incubated in primary antibody solution (anti-laminin, 1:50, Abcam, Germany) (anti-GFP 1:200, life technologies, Germany) (anti-dsRED, Clontech, Germany) (anti-pHH3, 1:100, Milipore, Germany) (anti-pSmad1/5/8, 1:25, Cell Signaling, Germany) in blocking solution. Samples were washed and incubated in secondary antibody solution (anti-rabbit Dylight, 1:300, anti-chicken Alexa 488, 1:300, Jackson, UK) with DAPI (stock: 2 µg/ml, 1:500) added. Consecutively, samples were washed and mounted for microscopy.
8
Analyzing Osteogenic Markers in Cultured Cells
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The cells were cultured under induction medium in a 12-well plate. Runx2, COL1A1, p-ERK and p-Smad1/5/9 were detected using fluorescence microscopy (Leica). Briefly, after 10 min of fixation at room temperature in 4% paraformaldehyde, the cells were blocked for 30 min in 0.04% Triton X-100 and 5% bovine serum albumin and then incubated overnight with anti RUNX2 (1:1,600; Cell Signaling Technology, Shanghai, China), anti-COL1A1 (1:500; Abcam, Cambridge, UK), anti-p-ERK (1:100; Cell Signaling Technology), anti-p-Smad1/5/8 (1:800; Cell Signaling Technology). They were then incubated with a fluorescence-conjugated secondary antibody (Beyotime, China) for 60 min. The cell nuclei were stained with DAPI (KeyGen Biotech, Nanjing, China) for 4 min. Cell immunofluorescence was observed using fluorescence microscopy (Leica, Germany).
9
Quantifying SMAD1/5/8 Phosphorylation
10
Protein Analysis of VSMCs
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VSMCs were lysed in 0.1 M Tris pH 8.1, 0.15 M NaCl, 1% triton x-100 0.2 mM NaVO3 and 1:50 protease inhibitor cocktail (Sigma). Protein concentrations were determined using DC protein assay (Bio-Rad, Veenendaal, the Netherlands) and lysates were separated on Any kD Mini-PROTEAN TGX Precast Protein Gels (BioRad). Samples were transferred to a PVDF or nitrocellulose membrane (BioRad) and incubated overnight with anti-αSMA (A2547, Sigma), anti-calponin (ab46794, Abcam), anti-ALP (R&D Systems, AF290), anti-Runx2 (MBL, D130-3), anti-β-catenin (BD, 610153), anti-p-SMAD1/5/8 (Cell Signalling, 12820P) and α-tubulin (Sigma, T6074) antibodies. Protein was then detected using HRP-conjugated secondary antibodies (anti-mouse: p0447, Dako; anti-rabbit: 7074S, Cell Signalling, anti-goat: P0449, Dako) and visualized by enhanced chemiluminescence (Pierce ECL Western Blotting Substrate, ThermoFisher Scientific).
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