Smad1 5 8

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Smad1/5/8 is a recombinant protein that functions as a regulator of gene transcription. It belongs to the Smad family of proteins, which are involved in the transforming growth factor-beta (TGF-β) signaling pathway.

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Lab products found in correlation

P smad1 5 8, by Cell Signaling Technology (7 mentions) Phospho smad1 5 8, by Cell Signaling Technology (4 mentions) Gapdh, by Santa Cruz Biotechnology (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Smad2 3, by Santa Cruz Biotechnology (2 mentions) P akt, by Cell Signaling Technology (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Anti rabbit hrp dab cell and tissue staining kit, by R&D Systems (1 mentions) Anti goat hrp dab cell and tissue staining kit, by R&D Systems (1 mentions) Nel741001kt, by PerkinElmer (1 mentions) Bmpr 2, by Santa Cruz Biotechnology (1 mentions) P smad2 3, by Santa Cruz Biotechnology (1 mentions) Ab13847, by Abcam (1 mentions) Phospho smad1 5 8, by Santa Cruz Biotechnology (1 mentions) Runx2, by Thermo Fisher Scientific (1 mentions) Osterix, by Santa Cruz Biotechnology (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Phospho smad2 3, by Cell Signaling Technology (1 mentions) Tris glycine gel, by Bio-Rad (1 mentions)

Close spelling variants of this product

Anti-SMAD1/5/8 Rabbit anti-Smad1/5/8 P-Smad1/5/8 Total SMAD1/5/8 Rabbit anti-p-smad1,5,8 Phospho-SMAD1/5/8 Anti-p-Smad1/5/8 Smad1/5/8 antibody

17 protocols using smad1 5 8

Immunohistochemical Analysis of Ovarian Development

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Ovaries derived from gilts at age of 30–400 days were fixed in 10% (w/v) paraformaldehyde with 0.02 MPBS (pH 7.2) at 4°C for about 2 h. Next, they were cut into vertical slices of about 0.5 cm thickness and fixed in fresh 10% (w/v) paraformaldehyde for a cumulative period of 24 h. These slices were mounted in paraffin, and serially cut into 5 μm-thick sections by Rotary Microtome (MICROM, Germany), and stained with HE. Ovarian HE sections were observed and photographed under a fluorescent microscope (Zeiss, Germany).
Immunohistochemistry detection was performed by using the anti-Rabbit HRP-DAB Cell and tissue staining kit (R&D, CTS005) and anti-Goat HRP-DAB Cell and tissue staining kit (R&D, CTS008). Immunohistofluorescence examination was performed by using TSA plus Fluorescein (PerkinElmer, NEL741001KT) and Cyanine3.5 (PerkinElmer, NEL763001KT) kit. Antibodies of BMP15 (Eterlife, EL806306-100), GDF9 (Eterlife, EL901942-100), FSHR (Eterlife, EL912710), luteinizing hormone receptor (LHR) (Eterlife, EL904141), Caspase3 (Abcam, ab13847), 3βHSD (Abcam, ab154385), p-Smad1/5 (CST, 9516), Ki67 (Abcam, ab15580) and LC3B (Arigo, ARG55799) were diluted 1:100 with PBS. Other antibodies including ALK6 (Santa Cruz, sc5679), BMPR2 (Santa Cruz, sc5683), Smad2/3 (Santa Cruz, sc8332), Smad1/5/8 (Santa Cruz, sc6031R), and p-Smad2/3 (Santa Cruz, sc11769) were diluted 1:50 in PBS.

Qin Y., Tang T., Li W., Liu Z., Yang X., Shi X., Sun G., Liu X., Wang M., Liang X., Cong P., Mo D., Liu X., Chen Y, & He Z. (2019). Bone Morphogenetic Protein 15 Knockdown Inhibits Porcine Ovarian Follicular Development and Ovulation. Frontiers in Cell and Developmental Biology, 7, 286.

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Bone Differentiation Pathway Modulation

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Culture materials and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). MAPK inhibitors including PD98059 (inhibitor for ERK), SP600125 (inhibitor for JNK), SB203580 (inhibitor for p38), and LDN193189 (inhibitor for ALK3) were purchased from Calbiochem (La Jolla, CA, USA). Antibodies against p38, phospho-p38, SMAD1/5/8, phospho-SMAD1/5/8, osterix, and OPN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody against Runx2 was purchased from Cell Signaling Technology (Beverly, MA, USA). The catalog numbers and dilutions for antibodies used in Western blot analysis were provided in Table S1. Neutralizing mABs against BMP-2, BMP-6 were purchased from R&D Systems (Minneapolis, MN, USA). Runx2, SMAD1 siRNA vectors, and a control siRNA construct (containing random DNA sequences) were purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals of reagent grade were obtained from Sigma (St. Louis, MO, USA).

Chen C.N., Chang H.I., Yen C.K., Liu W.L, & Huang K.Y. (2022). Mechanical Stretch Induced Osteogenesis on Human Annulus Fibrosus Cells through Upregulation of BMP-2/6 Heterodimer and Activation of P38 and SMAD1/5/8 Signaling Pathways. Cells, 11(16), 2600.

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Evaluation of BMP4 and Activin A Signaling

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iECs were plated on fibronectin following sorting at a density of 7.5 × 10⁴ cells per well of a 6-well plate and grown for 3 days in ECM medium (ScienCell). iECs were serum-starved for 1 h before a 40 min treatment with either 50 ng/ml of BMP4 or Activin A (R&D Systems). Cells were harvested in RIPA buffer (Pierce, Thermo Scientific) supplemented with 1X protease and a phosphatase inhibitor cocktail (Roche). Whole-cell lysates were prepared in Laemmli buffer (BioRad) and resolved in 4–20 % tris-glycine gels (BioRad). Primary antibodies towards SMAD1/5/8 (Santa Cruz Biotechnology), phospho-SMAD1/5/8 (Cell Signaling), SMAD2/3 (Cell Signaling), and phospho-SMAD2/3 (Cell Signaling) were used at a dilution of 1:1000. Anti-GAPDH antibody (Thermo Scientific) was used at a dilution of 1:10,000. Binding was visualized with horseradish peroxidase-conjugated antibodies (Cell Signaling) and ECL (Enhanced ChemiLuminescence) substrate (Thermo Scientific). An ImageQuant LAS 4000 (GE Healthcare) was used to image the blots and quantifications were done using Image J software.

Barruet E., Morales B.M., Lwin W., White M.P., Theodoris C.V., Kim H., Urrutia A., Wong S.A., Srivastava D, & Hsiao E.C. (2016). The ACVR1 R206H mutation found in fibrodysplasia ossificans progressiva increases human induced pluripotent stem cell-derived endothelial cell formation and collagen production through BMP-mediated SMAD1/5/8 signaling. Stem Cell Research & Therapy, 7, 115.

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Liver Nuclear Protein Analysis

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SDS–PAGE and western blot analysis were performed on liver nuclear extracts^{36 (link)}. Livers were removed, rinsed in ice-cold PBS, and used to prepare liver nuclear extracts with Nuclear Extract Kits (Active Motif, Carlsbad, CA). Nuclear protein extracts were separated by 10% SDS–PAGE gel and blotted onto nitrocellulose membranes (GE Healthcare, Little Chalfont, United Kingdom). The membranes were immunoblotted with the following antibodies: phospho-STAT3, STAT3, phospho-SMAD1/5/8 (Cell Signaling, catalogue #9511S, Danvers, MA), SMAD1/5/8 (Santa Cruz Biotechnology, catalogue #SC-6031-R, Santa Cruz, CA) and β-actin (Abcam, Cambridge, MA). As a secondary antibody, anti–rabbit IgG (Cell Signaling) or anti–mouse IgG (GE Healthcare) were used. Antigen–antibody complexes were visualized with the ECL Western Blotting Detection Reagent (GE Healthcare) in x-ray film. Films were digitalized using a scanner (HP psc2350, 300 dpi resolution), and obtained JPEG images were imported into Adobe Illustrator (300 dpi) and assembled.

Huang H., Zuzarte-Luis V., Fragoso G., Calvé A., Hoang T.A., Oliero M., Chabot-Roy G., Mullins-Dansereau V., Lesage S, & Santos M.M. (2020). Acute invariant NKT cell activation triggers an immune response that drives prominent changes in iron homeostasis. Scientific Reports, 10, 21026.

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Western Blot Analysis of Smad Phosphorylation

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Cell protein lysates were harvested from OCLs in modified RIPA buffer (50mM Tris pH 7.4, 150mM NaCl, 1% IGEPAL, 0.25% sodium deoxycholate, 1mM EDTA) supplemented with Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Scientific). Lysates were cleared by centrifugation at 12,000Xg at 4°C. Proteins were resolved by SDS-PAGE and transferred to a PVDF membrane (Millipore). Anti-FLAG (DYKDDDDK) antibody was obtained from Cell Signaling. Smad1/5/8 was obtained from Santa Cruz, and phospho-Smad1/5/8 (p-Smad1/5/8) antibody was obtained from Cell Signaling Technology. HRP-conjugated anti-rabbit or anti-mouse were incubated with membranes, washed, and incubated with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Typically, the membranes were blotted with p-Smad 1/5/8 before being reblotted with total Smad1/5/8 antibody. Blots were stripped with western blot membrane stripping buffer (100 mM glycine, pH 2.5, 200 mM NaCl, 0.1% Tween 20, 0.1% β-mercaptoethanol) for 5 minutes at 50°C. Densitometry analysis was performed on NIH Image J. Individual activation levels were determined by comparing each p-Smad 1/5/8 band to its total Smad 1/5/8 band, and relative expression was determined by comparing individual activation levels to control infected cell culture levels.

Huntley R., Davydova J., Petryk A., Billington CJ J.r., Jensen E.D., Mansky K.C, & Gopalakrishnan R. (2015). The Function of Twisted Gastrulation in Regulating Osteoclast Differentiation is Dependent on BMP Binding. Journal of cellular biochemistry, 116(10), 2239-2246.

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Molecular Interactions in Osteoblast Differentiation

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After the osteoblast precursor cells were cultured for the indicated times, the cells were washed with chilled PBS and lysed in extraction buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, PMSF, and protease inhibitors). For immunoprecipitation, 293T cells were transfected with Flag-Lmx1b and HA-Runx2 for 48 h, or mature osteoblasts were harvested after washing with ice-cold PBS and lysed in extraction buffer. The lysates were immunoprecipitated using the indicated antibodies. The cell lysates and precipitated samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) transferred to poly vinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The primary antibodies used included Runx2, p-Smad158 and Smad158 (Santa Cruz Biotechnology, Dallas, TX, USA); HA; Flag and Actin (Sigma); p-Akt, Akt, p-Erk, and Erk (Cell Signaling Technology, Danvers, MA, USA); and Alp, Osx, and Lmx1b (Abcam, Cambridge, UK). HRP-conjugated secondary antibodies (Abcam) were probed and developed with ECL solution (Millipore). Signals were detected using enhanced chemiluminescence and analyzed using the Azure c300 chemiluminescent western blot imaging system (Azure Biosystems Inc., Dublin, CA, USA).

Kim K., Kim J.H., Kim I., Seong S., Han J.E., Lee K.B., Koh J.T, & Kim N. (2022). Transcription Factor Lmx1b Negatively Regulates Osteoblast Differentiation and Bone Formation. International Journal of Molecular Sciences, 23(9), 5225.

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Antibody-based Protein Detection Assays

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Western blots and immunohistochemical staining were performed as described.²⁸, ²⁹, ³⁰ Antibodies used were SLC40A1 (NBP1‐21502, Novus Biologicals, Centennial, CO, USA, 1:500), GAPDH (sc‐32233, Santa Cruz Biotechnology, Dallas, TX, USA, 1:2000), AMPKα1 (2795, Cell Signaling Technology, Danvers, MA, USA, 1:1000 and sc‐19128, Santa Cruz Biotechnology, 1:500), AMPK‐α2 (2757, Cell Signaling Technology, 1:1000 and sc‐19131, Santa Cruz Biotechnology, 1:1000), pAMPK (Thr172, 2535, Cell Signaling Technology, 1:1000), Smad1/5/8 (sc‐6031, Santa Cruz Biotechnology, 1:1000), pSmad1/5/8 (9511, Cell Signaling Technology, 1:1000), HIF1α (14179, Cell Signaling Technology, 1:1000), Hydroxy‐HIF1α (Pro564) (3434, Cell Signaling Technology, 1:1000), pSer/Thr (ab17464, Abcam, 1:1000 ), PHD2 (4835, Cell Signaling Technology, 1:1000), HA (3724, Cell Signaling Technology, 1:1000) and His (12698, Cell Signaling Technology, 1:1000).

Wang C., Zhang W., Xu W., Liu Z, & Huang K. (2022). AMP‐activated protein kinase α1 phosphorylates PHD2 to maintain systemic iron homeostasis. Clinical and Translational Medicine, 12(5), e854.

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Western Blot Analysis of Osteogenic Signaling

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Western blot analysis was performed as previously described [65 (link)]. Briefly, proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and the membranes were blocked for 1 h at room temperature and incubated overnight at 4 °C with the primary antibodies as follows: AKT (1:1000, #4691, Cell Signaling Technology, Beverly, MA, USA), p-AKT (1:1000, #4060, Cell Signaling Technology), β−actin (1:1000, #sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BMP2 (CUSABIO, #CSB-PAO9419AORb, Houston, TX, USA), Smad1/5/8 (1 : 1000, #sc-6031-R, Santa Cruz Biotechnology), p-Smad1/5/8 (1:2000, 13820, Cell Signaling), GSK3β (1:1000, #12456, Cell Signaling), p-GSK3β (1:1000, #9336, Cell Signaling), RUNX2 (1:1000, #12556, Cell Signaling Technology), and Wnt3a (1:1000, #2721, Cell Signaling). Then, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. The protein signals were monitored in the ProteinSimple detection system (ProteinSimple Inc., Santa Clara, CA, USA).

Park K.R., Park J.E., Kim B., Kwon I.K., Hong J.T, & Yun H.M. (2021). Calycosin-7-O-β-Glucoside Isolated from Astragalus membranaceus Promotes Osteogenesis and Mineralization in Human Mesenchymal Stem Cells. International Journal of Molecular Sciences, 22(21), 11362.

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Fucoidan Regulation of MAPK and Smad Pathways

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Cells were seeded at a density of 1 × 10⁵ cells per 6-cm cell culture dish and grown in maintenance medium until confluence. They were then treated with fucoidan in the presence or absence of MAPK inhibitors. Cell cultures were harvested at the indicated times and extracted at 4 °C in PRO-PREP solution (Intron Biotechnology, Inc., Sungnam, Korea). Extracts were cleared by centrifugation, and protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). For western blots, 10 μg of each protein sample was separated by 10% SDS-polyacrylamide gel electrophoresis and electro-transferred onto nitrocellulose membranes. The blots were blocked with a buffer containing 0.05% Tween-20 and 3% bovine serum albumin and reacted sequentially with primary and secondary antibodies. Primary antibodies against phosphorylated JNK, BMP2 (Cell Signaling Technology, Beverly, MA, USA), ERK (GeneTex, Inc., Irvine, CA, USA), p38 MAP kinase and Smad 1/5/8 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were diluted 1:1000. Horseradish peroxidase-conjugated secondary antibodies were diluted 1:7000. Finally, the membranes were developed with enhanced chemiluminescence solution and exposed to X-ray film.

Kim B.S., Kang H.J., Park J.Y, & Lee J. (2015). Fucoidan promotes osteoblast differentiation via JNK- and ERK-dependent BMP2–Smad 1/5/8 signaling in human mesenchymal stem cells. Experimental & Molecular Medicine, 47(1), e128-.

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Western Blot Analysis of HIF-1α, BMP9, Smad1/5/8

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Cells or tissues were lysed in RIPA buffer (0.5% NP-40, 0.1% SDS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5). Proteins were separated by SDS–PAGE, transferred to PVDF membranes (Millipore), and probed overnight at 4 °C with primary antibodies directed against HIF-1α (1:1000; Abcam Inc., Toronto, ON, Canada), BMP9 (1:2000, G-23; Santa-Cruz Biotechnology, Dallas, TX, USA), phospho-Smad1/5/8 (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA), Smad1/5/8 (1:1000, H18; Santa-Cruz Biotechnology), GAPDH (1:1000, FL-335; Santa-Cruz Biotechnology), or β-actin (1:1000, AC-15; Sigma-Aldrich, Oakville, ON, Canada). Blots were washed in PBS-Tween and were incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (1:5000; Biorad). The protein bands were revealed on Amersham Hyperfilm™ ECL sheets (GE Healthcare, Burnaby, BC, Canada) according to the manufacturer’s instructions. The blots were digitized, and the bands were quantified by densitometry using ImageJ software v1.45s [32 ].

Drouin G., Couture V., Lauzon M.A., Balg F., Faucheux N, & Grenier G. (2019). Muscle injury-induced hypoxia alters the proliferation and differentiation potentials of muscle resident stromal cells. Skeletal Muscle, 9, 18.

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