Western blotting was carried out as previously described [27 (link)]. The primary antibodies used were anti-N-Cadherin (BD Biosciences, Breda, Netherlands #610920), anti-α-Smooth Muscle Actin (Sigma, Zwijndrecht, Netherlands #A2547), anti-Snail (Cell Signaling, Leiden, Netherlands #3879), anti-Smad2 (BD Biosciences, Breda, Netherlands #610842), anti-p-Smad2 (Cell Signaling, Leiden, Netherlands #3108), anti-Smad4 (Santa Cruz #sc7966), anti-TGFβRI (Santa Cruz, Heidelberg, Germany #sc 398), anti-TGFβRII (Santa Cruz, Heidelberg, Germany #sc-400), anti-Smad3 (Epitomics, Duiven, Netherlands #1735–1), anti-p-Smad3 (a kind gift from Dr Edward B Leof, Mayo Clinic, Rochester, Minnesota) and anti-β-actin (Sigma, Zwijndrecht, Netherlands #A5441). All the secondary antibodies were from Sigma, Zwijndrecht, Netherlands. Western quantification was performed using image J software.
Anti smad4
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-Smad4 is a specific antibody that recognizes the Smad4 protein, which is a key component of the transforming growth factor-beta (TGF-β) signaling pathway. This antibody can be used in various laboratory techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to detect and analyze the Smad4 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti smad2, by Cell Signaling Technology (5 mentions)
Anti smad3, by Cell Signaling Technology (4 mentions)
Anti gapdh, by Santa Cruz Biotechnology (4 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti smad2 3, by Cell Signaling Technology (3 mentions)
Anti c myc, by Santa Cruz Biotechnology (3 mentions)
Anti gapdh, by Merck Group (3 mentions)
Anti n cadherin, by BD (2 mentions)
Anti tgfβri, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti smad3, by Santa Cruz Biotechnology (2 mentions)
Anti α sma, by Abcam (2 mentions)
Anti p21cip1, by Santa Cruz Biotechnology (2 mentions)
Tgf β1, by R&D Systems (2 mentions)
Anti pten, by Cell Signaling Technology (2 mentions)
Anti p smad2 3, by Cell Signaling Technology (2 mentions)
Anti vimentin, by Santa Cruz Biotechnology (2 mentions)
Anti e cadherin, by Santa Cruz Biotechnology (2 mentions)
Anti phospho smad2, by Cell Signaling Technology (2 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (2 mentions)
31 protocols using anti smad4
1
Western Blotting of EMT Markers
2
Quantitative Western Blot Analysis of TGF-β Signaling
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ESCs and endometrial tissues were first lysed with lysis buffer and centrifuged at 12,000 × g for 15 min at 4°C. A BCA protein assay kit (Beyotime) was used to determine the quantity of protein. Protein samples (50 μg) were separated with SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% nonfat milk in TBST (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween-20, pH 7.4) for an hour at room temperature and then incubated in primary antibody overnight at 4°C. The primary antibodies used in this study were rabbit anti-β-actin (1 : 2500, control), anti-TGF-b1 (1 : 1000), anti-p-Smad2 (1 : 500), anti-Smad2 (1 : 1000), anti-p-Smad3 (1 : 1000), anti-Smad3 (1 : 500), anti-Smad4 (1 : 200), and anti-Smad7 (1 : 500) all from Santa Cruz Biotechnology. Membranes were then incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1 : 10,000) for an hour at room temperature. Protein bands were visualized using Luminata Crescendo Western HRP Substrate (Millipore, Billerica, MA, USA) and a molecular imager (Bio-Rad, Philadelphia, PA, USA). Densitometry analysis was determined relative to β-actin using 1-D Analysis Software (National Institutes of Health, USA).
3
Histological Analysis of Liver Tissue
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Paraffin-embedded and frozen sections of liver tissues were prepared as described previously [2 (link)]. For immunohistochemical analysis, paraffin-embedded sections were stained with hematoxylin and eosin (H&E) and Sirius Red, respectively. For immunofluorescence detection, paraffin sections were incubated with anti-Albumin (Affinity Biosciences, Cincinnati, OH, USA), anti-Smad4 (Santa Cruz Biotechnology, Shanghai, China), anti-α-SMA (Abcam, Cambridge, UK), anti-collagen I (Affinity Biosciences, Cincinnati, OH, USA), and anti-PCNA (Santa Cruz Biotechnology, Shanghai, China) primary antibodies, respectively; frozen sections were incubated with anti-F4/80, anti-CD11b, and anti-Gr-1 primary antibodies (BD Pharmingen, San Diego, CA, USA), respectively, and followed by incubation with Alexa Fluor 488- or 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). Cell nuclei were stained with DAPI. The results were evaluated under the microscope (DP71, OLYMPUS, Tokyo, Japan). Image J software (Image J 1.8.0, NIH, Bethesda, USA) was used to quantify the collagen deposition in Sirius Red staining and the positive areas in immunohistochemistry and immunofluorescence, which are presented in the form of percentage.
4
Investigating TGF-β1 Signaling Mechanisms
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TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Human has-miR-216b mimic and control miRNA mimic were from QIAGEN. Antibodies were purchased as follows: anti-Smad2 (Cat# L16D3) and anti-Smad3 (Cat# 9513S) from Cell Signaling; anti-p21CIP1 (Cat# sc-6246) and anti-Smad4 (Cat# sc-7966) from Santa Cruz Biotechnology; and anti-β-actin (Cat# A1978) from Sigma Biochemicals. Cigarette smoke condensate (CSC) was purchased from Murty Pharmaceuticals, Inc.
5
Immunostaining of Retinal Vascular Markers
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The following primary antibodies were used in the immunostaining: anti‐CD31 (Biocare Medical), anti‐SMA‐Cy3 (Sigma‐Aldrich Co.), and anti‐SMAD4 (Santa Cruz Biotechnology). For immunofluorescence of retinal vasculature, enucleated eyes were fixed in 4% paraformaldehyde, then retinas were isolated. Primary antibodies were applied in the retinas, then secondary antibodies conjugated with Alexa 405, 488, or 594 were used to detect primary antibodies. For immunohistochemistry, paraffin‐embedded tissues were sectioned and anti‐SMAD4 antibodies (Santa Cruz Biotechnology) were incubated on the rehydrated sections. Mouse‐on‐mouse AP polymer (Biocare Medical) was used as secondary antibodies according to the manufacturer's instruction. Images were captured using a BZ‐X700 microscope (Keyence).
6
Immunoprecipitation of SMAD4 Protein
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Cells were treated with BMP4 or TGFβ1 and lysed in 1 ml of nuclear extraction Buffer A for 15 min at 4 °C. After centrifugation, the nuclear pellets were lysed in hypotonic Buffer B for overnight at 4 °C. Equivalent amounts of nuclear proteins were diluted in immunoprecipitation buffer containing 40 mM Tris-Cl pH 7.4, 120 mM NaCl, 1 mM EDTA, 5 mM NaF, 10 mM β-glycerophosphate, 1 mM Na3VO4 and protease inhibitor cocktail and immunoprecipitated with 1 μg anti-SMAD4 (Santa Cruz Biotechnology, CA) for 16 h at 4 °C. After rocking, Protein G agarose beads (Sigma-Aldrich) were added and incubated for 4 h at 4 °C. Immunoprecipitates were pelleted by centrifugation at 200 g and washed three times with immunoprecipitation buffer. The bound proteins were analysed by western blotting. Information on the antibodies used in this assay are summarized in Supplementary Table 6 .
7
Immunohistochemical Analysis of Tumor Samples
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Tumor specimens were fixed in 10% formalin overnight and embedded in paraffin. For immunohistochemistry, slides were deparaffinized and rehydrated. Antigen retrieval was enhanced by boiling in a steam pot at pH 6 in Dako target retrieval solution (Dako) for 15 min, followed by cooling for 30 min and washing in distilled water. Nonspecific binding was blocked by using the Linaris avidin/biotin blocking kit (Vector Laboratories) according to the manufacturer’s instructions. Slides were incubated with primary antibodies for 30 min, rinsed in PBS-T (PBS with 0.5% Tween 20), incubated for 20 min with the appropriate secondary antibody using the Dako REAL detection system (Dako), and rinsed in PBS-T. After blocking of endogenous peroxidase and incubation with streptavidin-horseradish peroxidase (HRP) (20 min at room temperature), slides were developed with 3-amino-9-ethylcarbazole (AEC) (Dako) and counterstained with hematoxylin. All antibodies were diluted in Dako antibody diluent, including anti-SMAD4 (dilution 1:50; Santa Cruz), Ki67 (dilution 1:1,000, Sigma), and CK19 (dilution 1:200, Abcam).
8
Western Blot Analysis of SMAD4 and GAPDH
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Western Blot analysis was performed as previously described (29 (link)). Equal amounts of protein (30 μg) were loaded per lane. As primary antibodies anti-SMAD4 (diluted 1:500, Santa Cruz) and anti-GAPDH (diluted 1:5,000, Santa Cruz) antibodies were employed. For detection, an HRP-conjugated horse anti-mouse IgG was used as the secondary antibody (diluted 1:5,000; Cell signaling; Cell Signaling Technology, Danvers, MA, USA).
9
Western Blot Analysis of Cell Signaling
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Western blotting was performed as described previously [20 (link),21 (link)]. The following antibodies were used in this study: anti-SMAD4 (sc-7154 or sc-7966), anti-E-cadherin (sc-8426), anti-vimentin (sc-7557), anti-CD133 (sc-8304), anti-CD44 (sc-18849), anti-Sp1(sc-14027), anti-c-Jun (sc-1694), anti-Fos (sc-52), anti-Fast-1 (sc-377358), anti-Hes1 (sc-25392), anti-GAPDH (sc-32233; Santa Cruz Biotechnology, Inc.), anti-p-Akt (#4060), anti-Akt (#4691), anti-p-p44/42 (#9101),anti-p44/42 (#4695), anti-Pten (#9272), anti-NF-κB (#4764S), anti- EGFR (#4267), anti-p-EGFR tyr 992 (#2235), anti-p-EGFR tyr 1068 (#3777), anti-Smad2/3 (#5339), anti-p-Smad2/3 (#3101), anti-p-c-Jun (#2361; Cell Signaling Technology, Inc.), anti-Nestin (N5413), mouse anti-β-actin (Sigma- Aldrich Co.), anti-CD133/1 (AC133, Miltenyi Biotec.) and anti-TGF-β1 (ab9758, Abcam, Plc.).
10
ChIP-seq analysis of cardiac transcription factors
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E10.5 embryos were dissected, and the primary myocardium (AV canal/outflow tract) and atria and ventricle regions were separately collected in cold PBS. ChIP was performed with a True MicroChIP kit (Diagenode) according to the manufacturer’s protocol. Antibodies used for ChIP were anti-Gata4 (2 μg, Santa Cruz Biotechnology), anti-Smad4 (2 μg, Santa Cruz Biotechnology) and anti-H3K27ac (0.5 μg, Abcam ab4729). Chipped DNA were subjected to library preparation using the 5500 Series SOLiD Systems preparation kit (Applied Biosystems) according to manufactures recommendations and sequenced using the 5500 wildfire sequencer (SOLiD). Quantitative PCR was performed on a Roche LightCycler 480 System using Sybr Green detection. Fold enrichment indicates the ratio of ChIPed DNA to negative control regions, normalized for input DNA. Primer sequences are provided in Supplementary Information .
Hey2 ChIP was performed in CM7/1 mouse ESC with doxycycline-inducible Flag-Hey2 expression55 (link). 10^8 cells were induced with 50 ng μl−1 doxycycline for 48 h and ChIP was performed with an anti-Flag-M2 antibody (10 μg Sigma-Aldrich). Chipped DNA were subjected to library preparation using the NEBNext ChIP-seq sample preparation kit (New England Biolabs) according to manufacturer’s recommendations and sequenced on a GAIIx platform (Illumina).
Hey2 ChIP was performed in CM7/1 mouse ESC with doxycycline-inducible Flag-Hey2 expression55 (link). 10^8 cells were induced with 50 ng μl−1 doxycycline for 48 h and ChIP was performed with an anti-Flag-M2 antibody (10 μg Sigma-Aldrich). Chipped DNA were subjected to library preparation using the NEBNext ChIP-seq sample preparation kit (New England Biolabs) according to manufacturer’s recommendations and sequenced on a GAIIx platform (Illumina).
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