Smad3

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Smad3 is a protein that plays a key role in the transforming growth factor-beta (TGF-β) signaling pathway. It serves as an intracellular messenger, transmitting signals from the cell surface to the nucleus. Smad3 is involved in the regulation of various cellular processes, including cell growth, differentiation, and apoptosis.

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32 protocols using smad3

Western Blot Analysis of Cardiac Proteins

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Protein samples were isolated from the left ventricular myocardium of rats. Left ventricular myocardium lysates were prepared by homogenization in cell lysis buffer (Beyotime Institute of Biotechnology). The protein concentration was determined using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). The protein samples (40/20 µg) were mixed with 2X sodium dodecyl sulfate sample loading buffer (Beyotime Institute of Biotechnology) and were subsequently separated on a 12% polyacrylamide gel and blotted on a nitrocellulose membrane (Beyotime Institute of Biotechnology, Inc). The membranes were blocked with 5% non-fat milk, followed by incubation (at 4°C for 24 h) with antibodies specific for TGF-β1 (1:100, sc-146), TAK1 (1:100, sc-7162), Smad3 (1:100, sc-169248), Smad7 (1:100, sc-100140) or β-actin (1:100; sc-47778) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The membranes were subsequently incubated at 37°C for 30 min with horseradish peroxidase-conjugated goat anti-rabbit (cat. no. ZDR-5306), rabbit anti-goat (cat. no. ZDR-5308) and goat anti-mouse (cat. no. ZDR-5307) immunoglobulin G (1:1,000; all from Zhongshan Goldenbridge Biotechnology Corporation, Beijing, China) and enhanced chemiluminescence detection system (Bio-Rad Laboratories, Hercules, CA, USA) was used for visualization. The grey value was measured using Quantity One software.

XIAO X., CHANG G., LIU J., SUN G., LIU L., QIN S, & ZHANG D. (2016). Simvastatin ameliorates ventricular remodeling via the TGF-β1 signaling pathway in rats following myocardial infarction. Molecular Medicine Reports, 13(6), 5093-5101.

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Western Blot Analysis of SMAD Signaling

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Cells were lysed using protein extraction buffer at 48 h after transfection, and protein concentration was quantitated by BCA protein assay. Lysates (30 µg) were resolved by SDS/PAGE and transferred onto nitrocellulose membrane (Pall corporation, Port Washington, NY, USA). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): SMAD2 (catalogue no. 5339); phospho‐SMAD2 (catalogue no. 3108); SMAD3 (catalogue no. 9523); p21 (catalogue no. 2947); Santa Cruz Biotechnology (Delaware, CA, USA): SMURF2 (catalogue no. sc‐25511); Na⁺/K⁺ ATPase α (catalogue no. sc‐48345), (AbFrontier, Seoul, Korea): anti‐β‐actin (catalogue no. LFPA0207), (Sigma‐Aldrich, St. Louis, MO, USA): anti‐HA (catalogue no. H6908); anti‐Flag (catalogue no. F1804), (Abcam, Cambridge, UK): anti‐TβRI (catalogue no. ab31013); phospho‐SMAD3 (catalogue no. ab52903). Western Blotting Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to detect protein according to the manufacturer’s instructions. The membranes were visualized with an ATTO image analyzer (ATTO, Tokyo, Japan).

Chae D., Park J., Cho M., Ban E., Jang M., Yoo Y.S., Kim E.E., Baik J, & Song E.J. (2019). MiR‐195 and miR‐497 suppress tumorigenesis in lung cancer by inhibiting SMURF2‐induced TGF‐β receptor I ubiquitination. Molecular Oncology, 13(12), 2663-2678.

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Probing Cell Signaling Pathways with Western Blot

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Antibodies used for western blotting include TGFBR2 (Santa Cruz #sc-400, 1:1000), SMAD3 (Santa Cruz #sc-8332, 1:1000), SMAD4 (Santa Cruz #sc-1909-R, 1:1000), phosphor-SMAD3 (Cell Signaling Technology #9520, 1:2000), and GAPDH (ORIGENE #TA802519, 1:5000). The method of western blot was described in detail in ref. ^{22 (link)}.

Du X., Pan Z., Li Q., Liu H, & Li Q. (2018). SMAD4 feedback regulates the canonical TGF-β signaling pathway to control granulosa cell apoptosis. Cell Death & Disease, 9(2), 151.

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Immunohistochemical Analysis of IL-20 and Smad3

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Wax-embedded sections (4-μm thickness) were dewaxed in xylene and rehydrated in graded alcohols. For the immunostaining of IL-20 and Smad3, antigens were retrieved by incubation with citrate buffer (pH 6) for 9 minutes with a microwave. Endogenous peroxidase activity was inhibited with a solution of 0.3% H₂O₂ in methyl alcohol, after which sections were blocked with 5% donkey serum for 20 minutes and then reacted with antibodies to IL-20 (Abcam) or Smad3 (Santa Cruz Biotechnology) at 4°C. After excess antibody was washed out with PBS, samples were incubated with horseradish peroxidase (HRP)–labeled anti-mouse antibody (Nichirei Biosciences) at 37°C.
For immunostaining of IL-20R or Fli-1, antigens were retrieved by incubation with citrate buffer (pH 9) for 9 minutes with a microwave. Antibodies to IL-20R (Abcam) and Fli-1 (Santa Cruz Biotechnology) and HRP-labeled anti-rabbit antibody (Nichirei Biosciences) were used.
The reaction was visualized using a diaminobenzidine substrate system (Dojin). Slides were counterstained with Mayer’s hematoxylin and examined under a light microscope (BX50; Olympus).

Kudo H., Jinnin M., Asano Y., Trojanowska M., Nakayama W., Inoue K., Honda N., Kajihara I., Makino K., Fukushima S, & Ihn H. (2014). Decreased Interleukin-20 Expression in Scleroderma Skin Contributes to Cutaneous Fibrosis. Arthritis & rheumatology (Hoboken, N.J.), 66(6), 1636-1647.

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Protein Expression Analysis by Western Blot

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Proteins were extracted, separated by 10% polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. After transfer, the membranes were blocked in 5% bovine serum albumin for 2 h at room temperature, and incubated overnight at 4°C with primary antibodies (SMAD3, CDK4, bcl-2, COL1 and GAPDH) (Santa Cruz, USA), followed by 1 h incubation with a secondary antibody (Santa Cruz, USA). Proteins were quantified by densitometry, and normalized to GAPDH.

Wang H., Guo B., Hui Q., Lin F, & Tao K. (2020). CO2 lattice laser reverses skin aging caused by UVB. Aging (Albany NY), 12(8), 7056-7065.

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Western Blot Analysis of Smad Proteins

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The lysates were clarified by centrifugation and the supernatants collected. Protein concentrations were determined using the bicinchoninic acid assay (BCA) Protein Assay (Applygen, Beijing, China). Equivalent amounts of tissue protein (80 μg) were resolved on SDS polyacrylamide gels, and transferred by electroblotting to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% (W/V) nonfat milk at room temperature for 1 h, and then incubated overnight at 4°C with the primary antibody against Smad3 (dilution 1: 200, Santa Cruz, CA, USA), Smad7 (dilution 1:200, Santa Cruz, CA, USA), p-Smad3 (dilution 1:1000, Epitomics, CA, USA), and β-actin (dilution 1: 1000, Santa Cruz, CA, USA). After washing in a buffer containing Tris-buffered saline (TBS), with 0.1% Tween, the membranes were incubated with horseradish peroxidase (HRP)-linked anti-mouse secondary antibody at a dilution of 1:3000. Following washing in 0.1% Tween TBS buffer, the immunolabeled proteins were detected by enhanced chemiluminescense reagents (Applygen, Beijing, China). The density of the detected bands was analyzed by Quantity One software.

Nie Y., Li S., Yi Y., Su W., Chai X., Jia D, & Wang Q. (2014). Effects of astragalus injection on the TGFβ/Smad pathway in the kidney in type 2 diabetic mice. BMC Complementary and Alternative Medicine, 14, 148.

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Protein Extraction and Western Blotting

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Proteins were extracted from mouse hearts with T-PER tissue protein extraction reagent (Thermo Scientific) plus proteinase inhibitors. RIPA buffer plus proteinase inhibitors was used to extract protein from cardiomyocytes and cardiac fibroblasts isolated from adult mouse hearts and in vitro cultured NRVMs. Protein samples were quantified and separated with SDS-PAGE before transfer to a nitrocellulose membrane, followed by antibody probing. ChIP assays were performed as described previously^{3 (link)} with specific gene primers as indicated (listed in Supplementary Table 1). The following primary antibodies were used: GAPDH (Santa Cruz, sc-20358), H3K9Me2 (Abcam, ab1220), H3K9me3 (Abcam, ab8898), H3K4me3(Active Motif, 39159), and H3K27me3 (Millpore, 07-449), Histone H3(CellSignaling, 4499), FLAG (Sigma-Aldrich, F1804), TIMP1 (Invitrogen, MA1-773), ANP (Novus, NBP2-14873), TGFbeta1 (Abcam, ab-64715), p SMAD3 (Santa Cruz, sc11769), SMAD3(Santa Cruz, sc-133098). All antibodies were diluted 1:1000 in western blot. The original un-cropped western blots with molecular weight markers were presented in supplementary figure 7.

Zhang Q.J., Tran T.A., Wang M., Ranek M.J., Kokkonen-Simon K.M., Gao J., Luo X., Tan W., Kyrychenko V., Liao L., Xu J., Hill J.A., Olson E.N., Kass D.A., Martinez E.D, & Liu Z.P. (2018). Histone lysine dimethyl-demethylase KDM3A controls pathological cardiac hypertrophy and fibrosis. Nature Communications, 9, 5230.

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Quantitative Protein Expression Analysis

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Total proteins of tumour or normal (the skin of the same mouse) tissues were extracted by chilled RIPA lysis buffer (Pierce) and then subjected to the western blotting analysis with primary antibodies against CD31, VEGF, MMP-2, MMP-9, MMP-13, CXCR4, NKp46, p-Smad3, Smad3 (all from Santa Cruz Biotechnology) and E4BP4 (Cell Signaling) in 1:1,000, followed by incubation with the corresponding IRDyeTM800-conjugated secondary antibodies (1:10,000, Rockland Immunochemicals). β-Actin was used as an internal control. Expression levels of the proteins were detected by using LiCor/Odyssey infrared image system (LI-COR; Biosciences), and the band intensities were quantified with the Image J software (version 1.48, NIH, Bethesda). Images have been cropped for presentation; their full size images are presented in Supplementary Figs 15–20.

Tang P.M., Zhou S., Meng X.M., Wang Q.M., Li C.J., Lian G.Y., Huang X.R., Tang Y.J., Guan X.Y., Yan B.P., To K.F, & Lan H.Y. (2017). Smad3 promotes cancer progression by inhibiting E4BP4-mediated NK cell development. Nature Communications, 8, 14677.

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Western Blot Analysis of TGF-β1, Smad3, and HDAC4

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Frozen muscle samples were homogenized in Western blot lysis buffer (Beyotime, China) and centrifuged at 12,000×g for 5 min at 4°C. The total protein concentration of the supernatant was determined (BioRad, Hercules, CA, USA). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride membranes, blocked with 5% milk powder, and incubated with primary antibodies against TGF-β1 (Santa Cruz Biotechnology), Smad3 (Santa Cruz Biotechnology), and HDAC4 (Abcam, Cambridge, UK) at 4°C overnight and. with horseradish peroxidase-conjugated anti-rat IgG secondary antibody (60 min, room temperature). Signals were detected using a chemiluminescence system (Pierce Biotechnology, Rockford, IL, USA) and quantified using an image analysis system (Bio-Rad).

Huang Q.K., Qiao H.Y., Fu M.H., Li G., Li W.B., Chen Z., Wei J, & Liang B.S. (2016). MiR-206 Attenuates Denervation-Induced Skeletal Muscle Atrophy in Rats Through Regulation of Satellite Cell Differentiation via TGF-β1, Smad3, and HDAC4 Signaling. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 1161-1170.

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Western Blot Analysis of Cardiac Proteins

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Total proteins from the left ventricle was isolated using RIPA lysis buffer, and Western blot was conducted as described before.⁶, ¹¹, ¹² Briefly, the membranes were blocked with 3% BSA, followed by incubation at 4°C for overnight with indicated primary antibodies including phospho‐NF‐κB/p65 (ser276), phospho‐IκBα (ser32), IκBα, phospho‐Smad3 (Cell Signaling Technology), Smad3, Smad7, Smurf2, NF‐κB/p65, β‐actin (Santa Cruz Biotechnology), collagen I and III (Southern Biotech), and α‐SMA (Sigma). Subsequently, the membranes were incubated with IRDye 800‐conjugated secondary antibody (Rockland Immunochemicals) and photographed with Odyssey infrared image system (LI‐COR Biosciences). Finally, the intensity of the bands was calculated by the Image J software and normalized against GAPDH.⁶, ¹¹, ¹²

Dong L., Li J., Hu Z., Huang X., Wang L., Wang H., Ma R.C., Lan H, & Yang S. (2021). Deletion of Smad3 protects against diabetic myocardiopathy in db/db mice. Journal of Cellular and Molecular Medicine, 25(10), 4860-4869.

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