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Rabbit anti smad3

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Rabbit anti-Smad3 is a primary antibody that specifically recognizes the Smad3 protein. Smad3 is a member of the SMAD family of signal transducer proteins that play a role in the transforming growth factor-beta (TGF-β) signaling pathway.

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Lab products found in correlation

Rabbit anti p smad3, by Cell Signaling Technology (4 mentions) Mouse anti rabbit igg hrp, by Santa Cruz Biotechnology (2 mentions) Mouse anti gapdh, by Santa Cruz Biotechnology (2 mentions) Rabbit anti mmp13, by Abcam (2 mentions) Mouse anti active β catenin, by Merck Group (2 mentions) Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (2 mentions) Mouse anti cbfβ, by Santa Cruz Biotechnology (2 mentions) Rabbit anti yap, by Santa Cruz Biotechnology (2 mentions) Mouse anti β actin, by Merck Group (2 mentions) Rabbit anti psmad3, by Abcam (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Rabbit anti six2, by Proteintech (1 mentions) Goat anti rabbit immunoglobulin g igg, by Proteintech (1 mentions) Mouse anti β tubulin, by Cell Signaling Technology (1 mentions) Goat anti mouse igg, by Proteintech (1 mentions) D glucose, by Merck Group (1 mentions) Rabbit anti nf κb p65, by Cell Signaling Technology (1 mentions) Creatinine assay kit, by Nanjing Jiancheng (1 mentions) Albumin elisa kit, by Abcam (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Smad3 (242 citations) Anti-Smad3 (93 citations) Rabbit anti phospho-Smad3 (7 citations) Rabbit anti-p-Smad3 (7 citations) Rabbit anti-Smad3 antibody (4 citations) Rabbit anti-phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (3 citations) Rabbit monoclonal anti-Smad3 (phosphor C25A9) (3 citations) Rabbit anti-phospho-Smad3 (ser423/425), (2 citations)

15 protocols using rabbit anti smad3

1

Protein Expression Analysis in mK3 Cells

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The mK3 cells were collected 48 h after the transfection. The proteins were extracted following the process as previously described [18 (link)]. The membranes were incubated with primary antibodies (mouse anti-β-Tubulin (1:2000 dilution, Cell Signaling Technology, Beverly, MA, USA); rabbit anti-Six2 (1:1000 dilution, Proteintech, Wuhan, China); rabbit anti-TβRII (1:300 dilution, Boster, Wuhan, China); rabbit anti-TβRI (1:500 dilution, Proteintech); rabbit anti-Smad3 (Cell Signaling Technology); rabbit anti-p-Smad3 (Abcam, Cambridge, MA, USA) at 4 °C overnight. The secondary antibodies were goat anti-rabbit Immunoglobulin G (IgG) and goat anti-mouse IgG (1:5000 dilution, Proteintech). The final signals were developed by Chemiluminescent Horseradish Peroxidase (HRP) Substrate Reagent (Millipore, Billerica, MA, USA) and were detected with ChemiDoc™ XRS+ (Bio-Rad, Hercules, CA, USA).
Mao Z., Lyu Z., Huang L., Zhou Q, & Weng Y. (2017). TβRII Regulates the Proliferation of Metanephric Mesenchyme Cells through Six2 In Vitro. International Journal of Molecular Sciences, 18(4), 853.
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2

Antibodies, Reagents, and Materials

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Antibodies were purchased from commercial sources, including rabbit anti-collagen IV from Proteintech (Wuhan, China), mouse anti-glyceraldehyde-3phosphate dehydrogenase (GAPDH) from Novus Biological (Littleton, CO, USA), rabbit anti-fibronectin and rabbit anti-TGF-β1 from Abcam (Cambridge, MA, USA), and rabbit anti-NF-κB-p65, rabbit anti-NF-κB-p-p65, rabbit anti-p-Smad3, and rabbit anti-Smad3 from Cell Signaling Technology (Beverly, MA, USA). Fetal bovine serum (FBS) was procured from Wisent Bioproducts (Saint-Bruno, QC, Canada), D-glucose, D-mannitol and Streptozotocin (STZ) from Sigma-Aldrich (Saint Louis, MO, USA), and Trizol from Life Technologies (Carlsbad, CA, USA), dulbecco’s modified eagle medium (DMEM) from Thermo Fisher Scientific (Waltham, MA, USA). Albumin ELISA kit was purchased from Abcam (Cambridge, USA), and creatinine assay kit was from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Wogonin was obtained from Topscience (Shanghai, China); Horseradish-peroxidase (HRP)-conjugated goat anti-rabbit/mouse IgG and 3.3-diaminobenzidine (DAB) was from Beijing Zhongshan Biotechnology (Beijing, China).
Zheng Z.C., Zhu W., Lei L., Liu X.Q, & Wu Y.G. (2020). Wogonin Ameliorates Renal Inflammation and Fibrosis by Inhibiting NF-κB and TGF-β1/Smad3 Signaling Pathways in Diabetic Nephropathy. Drug Design, Development and Therapy, 14, 4135-4148.
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3

Protein Interaction Analysis by Co-Immunoprecipitation

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Co-immunoprecipitations and immunoblot analyses were performed as previously described (70 (link)). Subcellular fractionation for co-immunoprecipitations on chromatin fractions was performed using the Subcellular Protein Fractionation Kit (Thermo Scientific, #78840) according to the manufacturer’s instructions. The primary antibodies used were as follows: mouse anti-p21 (Santa Cruz, sc-53870; 1:8000 used for both mouse and human samples), rabbit anti-Myc-tag (Cell Signaling, #2272; 1:1000); rabbit anti-Rb (Abcam, ab181616; 1:2000), rabbit anti-STAT1 (Abcam, ab92506; 1:1000), rabbit anti-STAT6 (Cell Signaling, #5397; 1:1000), rabbit anti-SMAD2 (Cell Signaling, #5339, 1:1000), rabbit anti-SMAD3 (Cell Signaling, #9513; 1:1000), and mouse anti-p27 (BD Biosciences, #610242, 1:1000). All antibodies were detected with secondary HRP-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson Immunoresearch; 1:10,000). PonS staining (0.2% w/v in 5% glacial acetic acid, Sigma-Aldrich, #P3504) served as a loading control. Immunoblots are representative of at least two independent experiments.
Sturmlechner I., Zhang C., Sine C.C., van Deursen E.J., Jeganathan K.B., Hamada N., Grasic J., Friedman D., Stutchman J.T., Can I., Hamada M., Lim D.Y., Lee J.H., Ordog T., Laberge R.M., Shapiro V., Baker D.J., Li H, & van Deursen J.M. (2021). p21 produces a bioactive secretome that places stressed cells under immunosurveillance. Science (New York, N.Y.), 374(6567), eabb3420.
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4

Molecular Pathways in Fibrosis Regulation

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JQ1 was purchased from Tocris Bioscience (Bristol, UK). The recombinant human TGF-β1 was from PeproTech (Rocky Hill, NJ, USA). Antibodies used in Western blot and sources were as follows. The rabbit anti-Brd4 antibody was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-α-SMA, anti-fibronectin and anti-collagen IV antibodies were purchased from Abcam (Cambridge, UK). Rabbit anti-Smad3, anti-p-Smad3, anti-ERK1/2, anti-p-ERK1/2 and anti-Nox4 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The rabbit anti-GAPDH antibody was from Bioss (Beijing, China). DCFH-DA was from Beyotime Biotechnology (Jiangsu, China), Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit from Molecular Probes (Eugene, OR, USA), polyethylene glycol (PEG)–catalase and N-acetyl-cysteine (NAC) from Sigma-Aldrich (St. Louis, MO, USA), SIS3 from Cayman Chemical (Ann Arbor, MI, USA), and U0126 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Zhou B., Mu J., Gong Y., Lu C., Zhao Y., He T, & Qin Z. (2016). Brd4 inhibition attenuates unilateral ureteral obstruction-induced fibrosis by blocking TGF-β-mediated Nox4 expression. Redox Biology, 11, 390-402.
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5

Immunoprecipitation and Western Blot Analysis

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Briefly, cells were lysed in Nonidet P-40 buffer. Protein A-Sepharose beads (Santa Cruz Biotechnology) and 5 μg of primary antibody or control rabbit IgG was added to lysates and incubated at 4°C with constant rotation for 12 h. Following centrifugation, pelleted beads were washed five times with Nonidet P-40 buffer. The final pellets were resuspended in 20 μl of sample buffer and heated to 100°C for 5 min. Immunoprecipitated proteins were then separated on 10% SDS-PAGE, transferred to nitrocellulose membranes and detected by immunoblotting with the following primary antibodies: mouse anti-HIF-1α, rabbit anti-p-Smad3 and rabbit anti-Smad3 (Cell Signaling).
Shi X., Guo L.W., Seedial S.M., Si Y., Wang B., Takayama T., Suwanabol P.A., Ghosh S., DiRenzo D., Liu B, & Kent K.C. (2014). TGF-β/Smad3 inhibit vascular smooth muscle cell apoptosis through an autocrine signaling mechanism involving VEGF-A. Cell Death & Disease, 5(7), e1317-.
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6

Western Blot Analysis of Protein Expression

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Proteins were loaded on SDS-PAGE and electro-transferred on nitrocellulose membranes. Immunoblotting was performed according to the manufacturer’s instructions. The following primary antibodies were used: mouse-anti-Cbfβ (Santa Cruz Biotechnology Cat# sc-56751, RRID:AB_781871), rabbit-anti-MMP13 (Abcam Cat# ab39012, RRID:AB_776416), rabbit-anti-Yap (Santa Cruz Biotechnology Cat# sc-15407, RRID:AB_2273277), mouse-anti-GAPDH (Santa Cruz Biotechnology Cat# sc-365062, RRID:AB_10847862), mouse-anti-Active-β-catenin(Millipore Cat# 05–665, RRID:AB_309887), rabbit-anti-Smad3 (Cell Signaling Technology Cat# 9513, RRID:AB_2286450), and rabbit-anti-pSmad3 (Cell Signaling Technology Cat# 9520 (also 9520 S, 9520 P), RRID:AB_2193207). Secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology Cat# sc-2004, RRID:AB_631746), and rabbit anti-mouse IgG-HRP (Santa Cruz Biotechnology Cat# sc-358917, RRID:AB_10989253). Quantification of Western blot area was performed by ImageJ.
Chen W., Lu Y., Zhang Y., Wu J., McVicar A., Chen Y., Zhu S., Zhu G., Lu Y., Zhang J., McConnell M, & Li Y.P. (2024). Cbfβ regulates Wnt/β-catenin, Hippo/Yap, and Tgfβ signaling pathways in articular cartilage homeostasis and protects from ACLT surgery-induced osteoarthritis. eLife, 13, e95640.
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7

Immunohistochemical Analysis of Kidney Sections

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Obstructed and contralateral (CON) kidney sections were deparaffinized in xylene, rehydrated in ethanol, subjected to antigen retrieval in sodium citrate buffer with subsequent addition of TWEEN 20 and washed in water. Endogenous peroxidase activity was quenched in BLOXALL (Vector Labs, SP-6000) followed by blocking in 10% normal goat serum. Sections were overlayed for 30 minutes with rabbit anti-PAI-1 (1:1000), rabbit anti-p53 (Thermo Scientific, MA1-39548; 1:200) or rabbit anti-SMAD3 (Cell Signaling; 1:1000) antibodies in 1% bovine serum albumin followed by incubation with appropriate secondary biotinylated antibody (Vector Labs; anti-rabbit BA-1000, anti-mouse BA-9200) for 30-minutes. VECTASTAIN Elite ABC (Vector Labs, PK-7100) was added for 30 minutes and reactions developed with ImmPACT DAB (3,3′-diaminobenzidine) peroxidase substrate (Vector Labs, SK-4105) for 2 to 10-minutes (with timing titrated according to staining intensity) prior to counterstaining with Hematoxylin QS (Vector Labs, H-3404).
Overstreet J.M., Samarakoon R., Meldrum K.K, & Higgins P.J. (2014). Redox control of p53 in the transcriptional regulation of TGF-β1 target genes through SMAD cooperativity. Cellular signalling, 26(7), 1427-1436.
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8

Investigating TGF-β1-Induced Fibrosis Pathways

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Tetra were from Sigma-Aldrich, Co. (T1512, St. Louis, MO, USA). Pifithrin-α hydrobromide was from MedChemExpress (HY-15484, Monmouth Junction, NJ, USA). Recombinant Human TGF-β1 Protein was from R&D Systems, Inc. (Minneapolis, MN, USA). The following primary antibodies were used: rabbit anti‐TNAP (ab108337, Abcam, Cambridge, UK), rabbit anti‐α-SMA (ab124964, Abcam), rabbit anti‐Vimentin (ab92547, Abcam), rabbit anti‐Fibronectin (ab2413, Abcam), rabbit anti‐TGF-β1 (SAB4502954, Sigma-Aldrich, Co.), rabbit anti-p-Smad2 (Ser465/Ser467) (#18338, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Smad2 (#5339, Cell Signaling Technology), rabbit anti-Smad3 (#9523, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-AMPKα1/2 (phosphoThr183/172, YT0216, ImmunoWay Biotechnology, Co., Plano, TX, USA), rabbit anti-p-AMPKα1/2 (YP0575, ImmunoWay), rabbit anti-p53 (ab26, Abcam), rabbit anti-cyclinE (YT1176, ImmunoWay), and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (YM1038, ImmunoWay). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (RS0004) or anti-mouse (RS0001) were from ImmunoWay. Goat anti-rabbit IgG secondary antibodies Alexa Fluor® 488 (RS23220, ImmunoWay) were used for immunofluorescent analysis.
Gao L., Wang L.Y., Liu Z.Q., Jiang D., Wu S.Y., Guo Y.Q., Tao H.M., Sun M., You L.N., Qin S., Cheng X.C., Xie J.S., Chang G.L, & Zhang D.Y. (2020). TNAP inhibition attenuates cardiac fibrosis induced by myocardial infarction through deactivating TGF-β1/Smads and activating P53 signaling pathways. Cell Death & Disease, 11(1), 44.
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9

Phosphorylation Signaling Antibody Panel

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The primary antibodies used in this study include rabbit anti-phospho-Smad3Ser423/425 (#9520, 1:500; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Smad3 (#9513, 1:1000; Cell Signaling Technology), rabbit anti-phospho-ezrinThr567 (PA5-37763, 1:500; Invitrogen, Carlsbad, CA, USA), mouse anti-ezrin (#35-7300, 1:1000; Invitrogen), rabbit anti-phospho-PKA α/β CAT (pThr197) (SAB4301240, 1:500; Sigma-Aldrich, Burlington, MA, USA), rabbit anti-PKA (06-903, 1:800; Sigma-Aldrich), rabbit anti-phospho-PKA substrates antibody (#9624, 1:1000; Cell Signaling Technology), rabbit anti-NADPH oxidase 4 (ab133303, 1:750; Abcam, Cambridge, MA, USA), and mouse anti-GAPDH (G8795, 1:10,000; Sigma-Aldrich) antibodies.
Guo W., Gao H., Pan W., Yu P, & Che G. (2021). High glucose induces Nox4 expression and podocyte apoptosis through the Smad3/ezrin/PKA pathway. Biology Open, 10(5), bio055012.
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10

Western Blot Analysis of SMAD3, E-cadherin, and N-cadherin

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Protein was collected and extracted from A549 and H1299 cells with RIPA lysis buffer and protease inhibitor cocktail and protein phosphatase inhibitor. The samples were then transferred to PVDF membranes, following electrophoresis and the membranes were incubated with rabbit-anti SMAD3 (Cell Signaling Technology, Inc.) or mouse-anti E-cadherin or mouse-anti N-cadherin (both Santa Cruz Biotechnology, Inc.) primary antibodies(SC-8426 for E-cadherin, SC-8424 for N-cadherin) overnight at 4 °C. The following day, the membranes were incubated with indicated secondary antibodies (Santa Cruz Biotechnology, Inc, SC-2005) for 1 h at room temperature. Detection was performed using the electrochemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). β-actin (Santa Cruz Biotechnology, Inc.) was used as the internal control.
Zhang J.X., Yang W., Wu J.Z., Zhou C., Liu S., Shi H.B, & Zhou W.Z. (2021). MicroRNA-32-5p inhibits epithelial-mesenchymal transition and metastasis in lung adenocarcinoma by targeting SMAD family 3. Journal of Cancer, 12(8), 2258-2267.
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