Smad2

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Smad2 is a protein involved in the transforming growth factor-beta (TGF-β) signaling pathway. It functions as an intracellular signal transducer and transcriptional modulator, playing a role in the regulation of various cellular processes.

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Lab products found in correlation

Close spelling variants of this product

P-Smad2/3 (22 citations) P-Smad2 (12 citations) Anti-Smad2 (12 citations) Anti-p-Smad2/3 (11 citations) Anti-p-Smad2 (5 citations) Phospho-Smad2 (4 citations) Anti-phosphorylated Smad2/3 (4 citations) Smad2/3 (sc133098) (4 citations) Goat anti-human p-SMAD2/3 (Ser 423/425) pAb (3 citations) Anti-SMAD2/3 antibody (3 citations)

17 protocols using smad2

Immunocytochemistry and Immunohistochemistry of TGF-β Signaling

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For immunocytochemistry, serum-starved MEFs were grown on glass slides, treated with TGF-β1, fixed with cold 4% paraformaldehyde (PFA) for 15 min, and then incubated in NH₄Cl/PBS (50 mM) for 10 minutes. Cells were permeabilized with 0.25% Triton X-100, blocked in 3% BSA for 30 minutes at room temperature, and incubated with primary antibodies at 4°C overnight. Cells were immunolabeled with the following antibodies: Smad2 (Santa Cruz), Smad3 (Cell Signaling), α-SMA (Sigma), TGF-β RI (Santa Cruz), TGF-β RII (Santa Cruz), and EEA1 (BD Biosciences). FITC- or rhodamine-conjugated secondary antibodies were used, and nuclei were counterstained with Hoechst 33342 (Invitrogen). Mounted cells were analyzed by confocal laser scanning microscopy using a Zeiss LSM Meta 510. For immunohistochemistry, skin samples were fixed in 4% PFA, embedded in paraffin, and sectioned. Sections were heated in DAKO Target Retrieval Solution (pH 6.0) for antigen retrieval, blocked in 5% BSA for 1 hour at room temperature, and incubated with Smad2 (Santa Cruz) primary antibody at 4°C overnight. Samples were incubated with HRP-conjugated secondary antibody and exposed to DAB for colorimetric detection. Nuclei were counterstained with hematoxylin. Mounted samples were analyzed by digital fluorescent microscopy.

Chang H.M., Lin Y.Y., Tsai P.C., Liang C.T, & Yan Y.T. (2014). The FYVE Domain of Smad Anchor for Receptor Activation (SARA) Is Required to Prevent Skin Carcinogenesis, but Not in Mouse Development. PLoS ONE, 9(8), e105299.

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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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Autophagy and EMT Regulation Pathways

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Chloroquine (CQ), 3-methyladenine (3-MA), rapamycin and TGF- β₂ were purchased from Sigma-Aldrich (St Louis, MO, USA). LY2109761 was from Selleck Chemicals (Houston, TX, USA). Antibodies against microtubule-associated protein 1 light chain 3-I (LC3-I), LC3-II, p62, Beclin 1, ATG7, poly (ADP-ribose) polymerase (PARP), cleaved-PARP, caspase 9, cleaved-caspase 9, Bax, and Bcl-x1 were from Abcam (Cambridge, MA, USA). Antibodies against TGF-β₂, Smad2, p-Smad2, E-cadherin, α-catenin, β-catenin, N-cadherin, fibronectin, ZO-1, Vimentin, MMP-9, Snail, Slug and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies of LC3-II and E-cadherin and secondary antibodies Alexa Fluor 568 anti-mouse IgG and Alexa Flour 568 anti-rabbit antibodies were from Jackson Immuno Research (Lancaster, PA, USA).

Li Z., Lu C., Wang F., Guo H., Wang Z., Yin H, & Li J. (2023). Heat treatment-induced autophagy promotes breast cancer cell invasion and metastasis via TGF-β2-mediated epithelial-mesenchymal transitions. PeerJ, 11, e14640.

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Western Blot Analysis of GC Cell Proteins

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The proteins extracted from GC cells and tissues were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% non-fat powdered milk in Tris-buffered saline for 2 h, membranes were incubated at 4 °C overnight with the following specific primary antibodies: WWP1 (Sigma, 1:200), smad4, TGFβR (Cell Signaling Technology, 1:1,000), smad2, p15, p16, p21, p53, and GAPDH (Santa Cruz Biotechnology, 1:200). The membranes were then incubated with HRP-conjugated anti-mouse or anti-rabbit IgG (1:2,000) at room temperature for 2 h and then washed with TBST buffer three times. Protein expression levels were visualized using an enhanced chemiluminescence (ECL) detection system. GAPDH was used as an internal control.

Li Q., Li Z., Wei S., Wang W., Chen Z., Zhang L., Chen L., Li B., Sun G., Xu J., Li Q., Wang L., Xu Z., Xia Y., Zhang D., Xu H, & Xu Z. (2017). Overexpression of miR-584-5p inhibits proliferation and induces apoptosis by targeting WW domain-containing E3 ubiquitin protein ligase 1 in gastric cancer. Journal of Experimental & Clinical Cancer Research : CR, 36, 59.

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Protein Lysate Preparation and Western Blot Analysis

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Protein lysates were prepared as previously described in RIPA [65 (link)]. Western blot analysis was performed with antibodies specific for beta-actin, Flag (Sigma Aldrich), GAPDH (Life technologies), CR-1, E-cadherin (Epitomics), vimentin (Dako, Trappes, France), p-SRC, SRC, p-SMAD2, p-ERK1/2, ERK1/2, p-FGFR1-4, p-AKT, AKT (Cell Signaling), FGFR3, FGFR1, SMAD2 (Santa Cruz, CA, USA).

Terry S., El-Sayed I.Y., Destouches D., Maillé P., Nicolaiew N., Ploussard G., Semprez F., Pimpie C., Beltran H., Londono-Vallejo A., Allory Y., de la Taille A., Salomon D.S, & Vacherot F. (2015). CRIPTO overexpression promotes mesenchymal differentiation in prostate carcinoma cells through parallel regulation of AKT and FGFR activities. Oncotarget, 6(14), 11994-12008.

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Western Blot Analysis of EMT Markers

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For western blotting, MDCK cells were lysed in 300 μL of cell lysis buffer containing 150 mM NaCl, 1% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris (pH 8.0), and a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Whole cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk, the membranes were incubated with primary antibodies against E-cadherin (1:500; BD Biosciences, Bedford, UK), α-SMA (1:10000; R&D, Minneapolis, IL, USA), fibronectin (1:1000; Santa Cruz, CA, USA), phospho-p38 (1:1000; Santa Cruz), p38 (1:1000; Abcam, Cambridge, UK), phospho-Smad2 (1:500; Millipore), Smad2 (1:1000; Santa Cruz), phospho-Smad3 (1:500; Abcam), Smad3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), and GAPDH (1:1000; Cell Signaling Technology, Danvers, MA, USA). The membranes were then washed three times in 1× PBS with Tween-20 (VWR Amresco, Solon, Ohio, USA) for 5 min and incubated with horseradish peroxidase-conjugated secondary antibodies. Target proteins were visualized using Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences). Band densities were measured using NIH Image J software.

Lee M., Kim S.H., Jhee J.H., Kim T.Y., Choi H.Y., Kim H.J, & Park H.C. (2020). Microparticles derived from human erythropoietin mRNA-transfected mesenchymal stem cells inhibit epithelial-to-mesenchymal transition and ameliorate renal interstitial fibrosis. Stem Cell Research & Therapy, 11, 422.

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Resveratrol Modulates TGF-beta1 Signaling

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Resveratrol was purchased from Shanghai Standardization for the Traditional Research Center and dissolved in dimethyl sulfoxide (DMSO). Human TGF-beta1 was obtained from PeproTech (USA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) was purchased from Promega (Madison, WI, USA). QuicBlock^TM Blocking Buffer for Immunol Staining, Antifade Mounting Medium with DAPI, QuicBlock^TM Secondary Antibody Dilution Buffer for Immunofluorescence and QuicBlock^TM Primary Antibody Dilution Buffer for Immunol Staining were purchased from Beyotime (Shanghai, China). Antibodies against MMP-2, MMP-9, α-SMA, Smad2, Smad3, P-Smad2, P-Smad3, Snail1, and Slug were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fibronectin, P-PI3K, PI3K, P-AKT, AKT, E-cadherin, and vimentin antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). LY290042, SB431542, SP600125, PDTC, and PD98059 were purchased from Selleck Chemical (Houston, TX, USA). PDTC and PD98059 were purchased from MedChemExpresss (New Jersey, NJ, USA). IRDye^TM fluorescence antibodies were obtained from Li-Cor Bioscience (Lincoln, NE, USA).

Sun Y., Zhou Q.M., Lu Y.Y., Zhang H., Chen Q.L., Zhao M, & Su S.B. (2019). Resveratrol Inhibits the Migration and Metastasis of MDA-MB-231 Human Breast Cancer by Reversing TGF-β1-Induced Epithelial-Mesenchymal Transition. Molecules, 24(6), 1131.

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Investigating Molecular Mechanisms in MDA-MB-231 Cells

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MDA-MB-231 cell was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). DMEM were purchased from Gibico (Brooklyn, NY, USA); MTS purchased from Promega (Madison, WI, USA); Smad2 and Smad3 were purchased from Santa Cruz; Fibronectin, P-PI3K, PI3K, P38, P-AKT, AKT, E-cadherin, Vimentin, Notch1, Wnt and NF-kB were purchased from Cell Signaling Technology (Danvers, MA, USA); LY290042, SB431542, SP600125 and PD98059 PDTC were purchased from Selleck Chemical Company (Houston, TX, USA); SB203580, Cyclopamin, ETC-159 and DAPT were purchased from MedChemExpresss (Monmouth Junction, NJ, USA); HRP-labeled goat anti-rabbit and anti-mouse IgG were purchased from Beijing Boda Tektronix Biogene Technology Co., Ltd. (Beijing, China); Resveratrol (98% content) was purchased from Shanghai Traditional Chinese Medicine Standardization Research Center (Shianghai, China). Cisplatin was purchased from Qilu Pharmaceutical (Hainan, China). IRDye^TM Fluorescent Antibody was purchased from Li-Cor Bioscience (Lincoin, NE, USA); Ultrasensitive Chemiluminescence Detection Kit was purchased from Shanghai Ya Enzyme Co., Ltd. (Shanghai, China); Immunofluorescence staining blocking solution (Containing DAPI) was purchased from Shanghai Biyuntian Company (Shanghai, China).

Yang M.D., Sun Y., Zhou W.J., Xie X.Z., Zhou Q.M., Lu Y.Y, & Su S.B. (2021). Resveratrol Enhances Inhibition Effects of Cisplatin on Cell Migration and Invasion and Tumor Growth in Breast Cancer MDA-MB-231 Cell Models In Vivo and In Vitro. Molecules, 26(8), 2204.

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TGF-β signaling pathway protocol

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Mv1Lu and NMuMG cells were obtained from ATCC (Manassas, VA). Na [¹²⁵I] (17 Ci/mg) was obtained from ICN Biochemicals (Irvine, CA). DMEM, high molecular mass protein standards (myosin, 205 kDa; β-galactosidase, 116 kDa; phosphorylase, 97 kDa; bovine serum albumin, 66 kDa), chloramine-T, disuccinimidyl suberate (DSS) and other biochemical reagents were obtained from Sigma (St Louis, MO). TGF-β (TGF-β₁) was purchased from Austral Biologicals (San Ramon, CA). Rabbit polyclonal antibodies to caveolin-1 (N-20), early endosome antigen 1 (EEA1), hemagglutinin (HA) epitope [Tsukazaki et al., 1998 (link)], P-Smad2, Smad2, P-Erk1/2, Erk1/2, α-actin, P-JNK, JNK, P-p38, p38, α-tubulin, TβR-I (ALK-5) and TβR-II, and SB-505124 [DaCosta Byfield et al., 2004 (link)] were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). TGF-β peptide antagonist [Huang et al., 1997 (link); Huang and Huang, 2005 (link); Singer et al., 2009 (link)] was synthesized by C S Bio Co. (Menlo Park, CA). The luciferase assay system was obtained from Promega (Madison, WI). The TβR-II-HA plasmid was purchased from Addgene (Cambridge, MA). The COL1A2-luc plasmid was constructed as described [Poncelet et al., 1999 (link)]. The SBE4-luciferase reporter plasmid was constructed as described [Jonk et al., 1998 (link)].

Huang S.S., Chen C.L., Huang F.W., Hou W.H, & Huang J.S. (2016). DMSO Enhances TGF-β Activity by Recruiting the Type II TGF-β Receptor From Intracellular Vesicles to the Plasma Membrane. Journal of cellular biochemistry, 117(7), 1568-1579.

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Immunoblotting of Jak2V617F and myelofibrosis

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SET-2 and HEL cells were washed in PBS following treatment with the inhibitors and lysed in RIPA lysis buffer containing protease inhibitors. Jak2V617F mouse BM cells and MF BM and PBMC were lysed directly by boiling in 2x sample buffer. Immunoblotting was performed using indicated phospho-specific or total antibodies. The following antibodies were used. Cell Signaling Technology: p-STAT5 (Tyr694) (#4322), STAT5 (#94205), p-RB (Ser795) (#9301), p-p65 (Ser536) (#3033), p-SMAD2 (Ser465/Ser467) (#18338), SMAD2 (#5339), Santa Cruz Biotechnology: p65 (#sc-372), CDK6 (#sc-177), RB (#sc-50). Sigma: β-Actin (#A5441), Abcam: HMGA2 (#ab202387), Abclonal: AURKA (#A2121), BD Biosciences: AURKB (#611082).

Dutta A., Nath D., Yang Y., Le B.T, & Mohi G. (2021). CDK6 is a therapeutic target in myelofibrosis. Cancer research, 81(16), 4332-4345.

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