Smad4

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Germany, United Kingdom

SMAD4 is a protein that functions as a central mediator of the transforming growth factor-beta (TGF-β) signaling pathway. It is a transcription factor that regulates the expression of target genes in response to TGF-β signaling.

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Lab products found in correlation

Close spelling variants of this product

Anti-Smad4 (31 citations) Smad4 (B-8, (7 citations) Smad4 (clone B8, (5 citations) Anti-SMAD4 antibody (5 citations) Anti-Smad4 (B-8, (4 citations) Anti-Smad4 (sc-7966) (4 citations) Mouse anti-Smad4 (3 citations) Smad4 siRNA (3 citations) Anti-SMAD4 mouse monoclonal antibody (clone B-8; (3 citations) Smad4 monoclonal antibody (B-8) (2 citations)

54 protocols using smad4

Protein Extraction and Western Blot Analysis

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The total protein was extracted from the cells using the M-PER mammalian protein extraction reagent (Pierce, IL, USA) or from rat joint synovial tissues using the T-PER tissue protein extraction reagent (Pierce). Equal amounts of protein (25 μg per lane), estimated by a bicinchoninic acid (BCA) protein assay kit (Pierce), were loaded onto (11%) SDS-PAGE gels and transferred onto nitrocellulose membranes. The blots were probed with a monoclonal antibody against rat Smad4 (1:300), TGF-β (1:800), α-SMA (1:600), collagen I (1:800), collagen III (1:600), Lama1 (1:800), Timp1 (1:800) and beta actin (1:1200) (Santa Cruz, USA), followed by the secondary HRP-conjugated anti-mouse/rabbit antibody (Santa Cruz, USA). After washing, the bands were detected by chemiluminescence and imaged with X-ray films. beta actin was used as an endogenous reference for normalization.

Xue M., Gong S., Dai J., Chen G, & Hu J. (2016). The Treatment of Fibrosis of Joint Synovium and Frozen Shoulder by Smad4 Gene Silencing in Rats. PLoS ONE, 11(6), e0158093.

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Western Blot Analysis of Hypoxia Signaling

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Cells were lysed with ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP-40; 0.25% sodium deoxycholate, 1mM EDTA), supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of extracts (30μg) were then electrophoresed on a sodium dodecyl sulfate (SDS) polyacrylamide gel and electroblotted to nitrocellulose filter membranes (Millipore). Then, membranes were immersed in blocking buffer (5% degreased milk powder) for 1 h and incubated with antibodies against HIF-1α, HIF-2α (Novus Biologicals, Littleton, CO), Smad5, TATA-binding protein (TBP), α-tubulin, Bmpr2, Bmpr1a (ProteinTech Group, Chicago, IL), β-actin or Smad4 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C. They were then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson Immuno-Research, West Grove, PA) and the protein bands were visualized using the SuperSignal chemiluminescent detection module (Pierce).

Zeng Y., Liu H., Kang K., Wang Z., Hui G., Zhang X., Zhong J., Peng W., Ramchandran R., Usha Raj J, & Gou D. (2015). Hypoxia inducible factor-1 mediates expression of miR-322: potential role in proliferation and migration of pulmonary arterial smooth muscle cells. Scientific Reports, 5, 12098.

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Quantifying Intracellular Smad4 in Muscle Stem Cells

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Injured and contralateral uninjured TAs were digested as described above. SCs/MPs were identified by an alternate cell surface staining panel selecting for Integrin α7 and negatively selecting for CD31, CD45, and Sca1 (Biolegend #102420, #103132, #108127). Cell fixation, permeabilization, and intracellular staining of Smad4 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-7966 PE) using BD Fixation Buffer (#554655) and BD Phosflow Perm Buffer III (#558050) was carried out according to the BD Phosflow protocol. Analysis of cell surface and intraceullar staining was performed on an LSR II Flow Cytometer (BD Biosciences). Mean Fluorescent Intensity (MFI) was quantified with FlowJo software. Negative controls stained with all cell surface markers but not intracellular antibodies were analyzed to assess background staining. Negative control MFI was subtracted to obtain final MFI values.

Paris N.D., Soroka A., Klose A., Liu W, & Chakkalakal J.V. (2016). Smad4 restricts differentiation to promote expansion of satellite cell derived progenitors during skeletal muscle regeneration. eLife, 5, e19484.

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Western Blot Analysis of TGF-β Signaling Proteins

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BFTC-909 cells were lysed in RIPA buffer containing a protease inhibitor mixture (Roche, Basel, Switzerland). For each lane of 10% SDS–PAGE gel, 30 μg of cell lysate protein was loaded, separated, and subsequently transferred onto Immobilon-P Transfer Membrane (Millipore, Burlington, MA, USA). The membranes were probed with specific antibodies including the primary antibodies against INHBA (cat# sc-166503; Santa Cruz, CA, USA; 1:500), SMAD4 (cat# sc-7966; Santa Cruz, CA, USA; 1:1000), SMAD3 (cat# ab40854; Abcam, Cambridge, UK; 1:1000), SMAD2 (cat# ab40855; Abcam, Cambridge, UK; 1:1000), and beta-actin (cat# ZRB1312; Sigma, St. Louis, MO, USA; 1:5000). The secondary antibodies were added and incubated for 1 h and visualized using chemiluminescence. Enhanced chemiluminescence Western blotting reagents were obtained from Pierce Biotechnology (Rockford, IL, USA).

Kao C.C., Chang Y.L., Liu H.Y., Wu S.T., Meng E., Cha T.L., Sun G.H., Yu D.S, & Luo H.L. (2022). DNA Hypomethylation Is Associated with the Overexpression of INHBA in Upper Tract Urothelial Carcinoma. International Journal of Molecular Sciences, 23(4), 2072.

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Probing Cell Signaling Pathways with Western Blot

Cited 1 time

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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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Western Blot Analysis of Tumor Proteins

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Lysates were generated by incubating cells or ground tumors in RIPA buffer (Millipore). 20–30 μg of cleared lysate were analyzed by SDS-PAGE as previously described ^{11 (link)}. The following primary antibodies were used: Vinculin (1:5,000 dilution; Sigma, V9131), β-Actin (1:5,000; Sigma, A5441), Collagen I (1:500; Abcam, ab21286), Fibronectin (1:1,000; Abcam, ab2413), Smad4 (1:200; Santa Cruz, sc-7966), Smad2 p-S465/467 (1:1,000; Cell Signaling, 3108S), Smad2/3 (1:1,000; Cell Signaling, 3102S), GCN2 p-T899 (1:1,000; Abcam, ab75836), GCN2 (1:1,000; Cell Signaling, 3302S), ATF4 (1:200; Santa Cruz, sc-200), PC (1:1,000; Novus, NBP1–49536), S6K (1:1,000; Cell Signaling, 2708S), GLUL (1:1,000; Sigma, G2781), SMA (1:1,000; Millipore, CBL171). The following secondary antibodies were used: anti-rabbit HRP (1:5,000; GE, NA934V), anti-mouse HRP (1:5,000; GE, NA931). Quantification of band intensities of Collagen I relative to β-Actin or Vinculin in tumor allograft experiments was performed with Image Lab software v6.0 (Bio-Rad).

Schwörer S., Pavlova N.N., Cimino F.V., King B., Cai X., Sizemore G.M, & Thompson C.B. (2021). Fibroblast pyruvate carboxylase is required for collagen production in the tumor microenvironment. Nature metabolism, 3(11), 1484-1499.

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EMT Regulation by BMP Signaling Pathway

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The antibodies used in this study included those targeting β-actin (NB600-501, Novus), E-cadherin (#3195, Cell Signaling Technology for western blot; 20,874–1-AP, Proteintech for IHC staining), occludin (13,409–1-AP, Proteintech), N-cadherin (ab53519, Abcam), Vimentin (#550,513, BD), Slug (sc-166476, Santa Cruz Biotechnology), BMPR2 (ab78422, Abcam), BMP2 (A0231, ABclonal), SMAD5 (#9517, Cell Signaling Technology), SMAD1 (#6944, Cell Signaling Technology), SMAD4 (sc-7966, Santa Cruz Biotechnology), and phospho-SMAD1/5 (ser463/465) (#9516, Cell Signaling Technology). Reagents included rhBMP-2 (R&D system), puromycin (Sigma-Aldrich), and LDN193189 (Cayman).

Wu C.K., Wei M.T., Wu H.C., Wu C.L., Wu C.J., Liaw H, & Su W.P. (2022). BMP2 promotes lung adenocarcinoma metastasis through BMP receptor 2-mediated SMAD1/5 activation. Scientific Reports, 12, 16310.

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Immunohistochemical Analysis of Tumor Markers

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All selected tissue specimens were stained for MLH1 (1:50; BD Pharmingen, San Diego, CA, USA), p16 (1:400; ImmunoLogic, Duiven, The Netherlands), SMAD4 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p53 (1:2000; Neomarkers, Fremont, CA, USA), as described previously (Figure 1A) [15 (link)]. MLH1 deficiency was defined as a complete absence of nuclear MLH1 staining. The complete absence of SMAD4 or p16 staining was considered indicative of dysfunction of the TGF‐β pathway or p16, respectively. Dysfunction of p53 was defined as either strongly positive or absent p53 staining in ≥75% of the epithelial nuclei.

Bleijenberg A.G., IJspeert J.E., Mulder J.B., Drillenburg P., Stel H.V., Lodder E.M., Carvalho B., Jansen J., Meijer G., van Eeden S., Dekker E, & van Noesel C.J. (2022). The earliest events in BRAF ‐mutant colorectal cancer: exome sequencing of sessile serrated lesions with a tiny focus dysplasia or cancer reveals recurring mutations in two distinct progression pathways. The Journal of Pathology, 257(2), 239-249.

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Western Blot Analysis of TGF-β Signaling

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Cell lysates were analysed by SDS-PAGE using the following antibodies: PO₄-SMAD2 (Ser465/467) (rabbit polyclonal, #3101, Cell Signalling Technology [CST]), SMAD2 (mouse monoclonal, C16D3, CST), SMAD2/3 (mouse monoclonal, Clone 18, BD transduction Laboratories), SMAD4 (mouse monoclonal, B-8, Santa Cruz Biotechnology), TGFBR1 (rabbit polyclonal, V-22, Santa Cruz Biotechnology), CDKN1A (rabbit polyclonal, C19, Santa Cruz Biotechnology), RHOA (mouse monoclonal, 26C4, Santa Cruz Biotechnology), PO₄-SRC (Tyr416) (rabbit monoclonal, D49G4, CST), SRC (rabbit monoclonal, 36D10, CST), PO₄-p44/p42 MAPK (ERK1/2) (Thr202/Tyr404) (rabbit polyclonal, #9101, CST), p44/p42 MAPK (ERK1/2) (rabbit polyclonal, #9102, CST), β-actin (mouse monoclonal, AC-74, Sigma). Secondary HRP-conjugated antibodies (Dako) and enhanced chemiluminescence (GE Healthcare) was used to detect bound antibody.

Spender L.C., Ferguson G.J., Liu S., Cui C., Girotti M.R., Sibbet G., Higgs E.B., Shuttleworth M.K., Hamilton T., Lorigan P., Weller M., Vincent D.F., Sansom O.J., Frame M., Dijke P.T., Marais R, & Inman G.J. (2016). Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells. Oncotarget, 7(50), 81995-82012.

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Apoptotic and mTOR Signaling Pathways in Cancer Cells

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Cordycepin, taxol, cisplatin, etoposide, wortmannin, and methylthiazol tetrazolium (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Caspase inhibitors Z-VAD-FMK, Z-IETD-FMK, and Z-LEHD-FMK (which inactivate all caspases, caspase-8, or caspase-9, respectively) were purchased from R&D Systems (Minneapolis, MN). Antibodies against cleaved caspase-8 (Asp387), cleaved caspase-9 (Asp353), cleaved caspase-3 (Asp175), cleaved caspase-6 (Asp162), cleaved caspase-7 (Asp198), LC3 I/II, mTOR, phosphorylated mTOR (p-mTOR; Ser2448), AKT, p-AKT (Ser473), ERK, p-ERK (Thr202/Tyr204), JNK, p-JNK (Thr183/Tyr185), p38, p-p38 (Thr180/Tyr182), p53, and p-p53 (Ser15) were purchased from Cell Signaling (Beverly, MA). Antibodies against p21, p-p21 (Thr145), CDK2, TGFβ2, TGFβ3, and Smad4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against β-actin were purchased from Sigma-Aldrich (St. Louis, MO), and antibodies against PARP were purchased from Oncogene (San Diego, CA).

Pan B.S., Wang Y.K., Lai M.S., Mu Y.F, & Huang B.M. (2015). Cordycepin induced MA-10 mouse Leydig tumor cell apoptosis by regulating p38 MAPKs and PI3K/AKT signaling pathways. Scientific Reports, 5, 13372.

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