Phospho smad2

Manufactured by Merck Group

Sourced in United Kingdom, United States, Germany

Phospho-Smad2 is a antibody used in laboratory research to detect the phosphorylated form of the Smad2 protein. Smad2 is a key mediator in the TGF-beta signaling pathway. The antibody enables the identification and quantification of the activated, phosphorylated Smad2 protein in cellular samples.

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Lab products found in correlation

Phospho smad3, by Cell Signaling Technology (2 mentions) Smad3, by Cell Signaling Technology (2 mentions) Gapdh, by Cell Signaling Technology (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Actin, by Santa Cruz Biotechnology (1 mentions) Phospho smad2 3, by Cell Signaling Technology (1 mentions) Dna pkcs, by Cell Signaling Technology (1 mentions) Smad2 3, by Cell Signaling Technology (1 mentions) Stemspan sfem medium, by STEMCELL (1 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions) Sc 25336, by Santa Cruz Biotechnology (1 mentions) Vinculin, by Santa Cruz Biotechnology (1 mentions) Ab3849, by Merck Group (1 mentions) P erk, by Santa Cruz Biotechnology (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Itgb1, by Abcam (1 mentions) Axio scan z1, by Zeiss (1 mentions) Tgfbi, by Thermo Fisher Scientific (1 mentions) Ncl fib, by Leica Biosystems (1 mentions)

10 protocols using phospho smad2

Analyzing TGFβ and Irradiation Signaling

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Whole cell lysates were prepared using RIPA cell lysis buffer (9803, Cell Signaling) and 1 mM PMSF (8553s, Cell Signaling). Lin^- cells, cells were cultured for 24 h in StemSpan SFEM medium (09600, StemCell Technologies) containing 2% L-glutamine (25030-081GIBCO), 1% penicillin/streptomycin (15140–122, GIBCO), 100 ng/ml SCF (250-03-10UG, Peprotech) and 100 ng/ml TPO (315-14-10UG, Peprotech). Lin^- cells were exposed to TGFβ1 (5 ng/ml) or TGFβ3 (5 ng/ml) along with LSN3301240 (5 μM) during 2 h. Western blots were performed using the following antibodies SMAD2/3 (86855, Cell Signalling), phospho-SMAD2 (ab3849, Millipore), phospho-SMAD2/3 (8828s, Cell Signaling) and Vinculin (sc-25336, Santa Cruz) antibodies.
Bone marrow stromal cell lines were exposed to irradiation (5 or 10 Gy), MEK inhibitor PD0325901 (10 μM) or TGFβ inhibitor LSN3301240 (5 μM). Western blots were performed with the following antibodies DNA-PKcs, 53BP1, RAD51, phospho-SMAD3 (9502s, Cell Signaling Technology, Danvers, MA, USA), p-ERK and actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Antibodies for analysis of fetal liver were DNA-PKcs, 53BP1, pERK1/2, total ERK1/2, CD45 CD41 and Vinculin.

Rodríguez A., Epperly M., Filiatrault J., Velázquez M., Yang C., McQueen K., Sambel L.A., Nguyen H., Iyer D.R., Juárez U., Ayala-Zambrano C., Martignetti D.B., Frías S., Fisher R., Parmar K., Greenberger J.S, & D’Andrea A.D. (2022). TGFβ pathway is required for viable gestation of Fanconi anemia embryos. PLOS Genetics, 18(11), e1010459.

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Immunohistochemistry of Tumor Biomarkers

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Sections of formalin-fixed, paraffin-embedded tumors were used. Briefly, after antigen retrieval, the following primary antibodies were used: ITGAV (sc-376,156, Santa Cruz,); ITGB1 (AB3167, Abcam), ITGB6 (HPA023626, Atlas), CEACAM7 (LS-B13068, LSBio), TGFBR1 (PA5–98192, Thermo Scientific), phospho-SMAD2 (AB3849, Millipore), TGFBI (MA526731, Thermo Fisher Scientific), Twist (ab50581, Abcam), Ki 67 (M7240, Agilent); human-specific fibronectin (NCL-FIB, Leica Biosystems); fibronectin, cross-reacting with the fibronectin-equivalent protein in mouse (A0245, Dako); STAT1 (HPA000931, Sigma-Aldrich), HLA-DR (M0746, Agilent). Vectastain ABC kit (Vector) was used. Slides were scanned by Axio Scan Z1 (Zeiss). For quantification with Image J (NIH), images of four randomly chosen vision fields (not containing necrotic areas) were analyzed. If artifacts interfered with automated quantification by Image J, the staining was quantified by two experienced investigators: grade 0: no reaction or weak focal reaction; grade 1 intense focal or diffuse weak reaction; grade 2 moderate diffuse reaction; grade 3 for an intense diffuse reaction.

Kemper M., Schiecke A., Maar H., Nikulin S., Poloznikov A., Galatenko V., Tachezy M., Gebauer F., Lange T., Riecken K., Tonevitsky A., Aigner A., Izbicki J., Schumacher U, & Wicklein D. (2021). Integrin alpha-V is an important driver in pancreatic adenocarcinoma progression. Journal of Experimental & Clinical Cancer Research : CR, 40, 214.

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Western Blot Analysis of Key Signaling Proteins

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Protein isolation and western blot were performed as described previously. [12] Primary antibodies were as following: DACH1 (Proteintech); c-Myc, p21, phospho-Smad3, cyclinD1 (Cell signaling Technology); CDK4 (Affinity); phospho-Smad2 (Millipore); cyclinE1, CDK2, Smad2 and Smad3 (Bioworld Technology); β-Actin (Beyotime). The blots were visualized using enhanced chemiluminescence (Pierce Bioscience).

Wu L., Herman J.G., Brock M.V., Wu K., Mao G., Yan W., Nie Y., Liang H., Zhan Q., Li W, & Guo M. (2014). Silencing DACH1 Promotes Esophageal Cancer Growth by Inhibiting TGF-β Signaling. PLoS ONE, 9(4), e95509.

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Comprehensive Protein Analysis Protocol

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Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% (v/v) NP-40, 5 mM EDTA pH 8.0, 5 mM EGTA pH 8.0, 20 mM NaF, 0.1 μM PMSF, 0.1 μM NaVO₃, plus complete protease inhibitor cocktail (Roche)) and cellular lysates were separated by SDS–PAGE and transferred to PVDF membranes. The following antibodies were used for protein detection: p38α MAPK (1/1,000, Cell Signaling, #9228), phospho-p38 (Thr180/Tyr182, 1/1,000, Cell Signaling, #9215), phospho-c-Jun (Ser63, 1/1,000, Cell Signaling, #9161), c-Jun (1/1,000, Cell Signaling, #L70B11), phospho-JNK (Thr183/Tyr185, 1/1,000, Cell Signaling, #9255), JNK (1/1,000, Cell Signalling, #9258), phospho-Smad2 (Ser465/Ser467, 1/1,000, Millipore, #AB3849), Smad2 (1/1,000, Cell Signalling, #3103), human CXCL12/SDF-1 antibody (1/1,000, R&D Systems, #AF-310-NA), tubulin (Sigma). For detection, we used Alexa Fluor 680- (1/5,000, Molecular Probes) or Li-Cor IRDye 800- (Rockland) labelled antibodies with the Odyssey Infrared Imaging System (Li-Cor).

Ruiz E.J., Oeztuerk-Winder F, & Ventura J.J. (2014). A paracrine network regulates the cross-talk between human lung stem cells and the stroma. Nature Communications, 5, 3175.

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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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Oxidative Stress and Fibrosis Markers

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Nox2/gp91phox (1:250 for WB, 1:100 IHC, BD Biosciences, San Diego, CA, 611414), Nox4 (1:500, Santa Cruz, Dallas, TX, sc-21860), α-SMA (1:2000 for WB, 1:50,000 for IHC, Sigma, St. Louis, MO, A2547), HNE (1:1000, Abcam, Cambridge, MA, ab46545), GAPDH (1:5000, Abcam, ab8245), Vimentin (1:500, Bioss, Woburn, MA, bs-0756R), Beta Actin (1:7500, US Biological, Salem, MA), Fibronectin (1:400, Abcam, ab23750), Phospho-Smad2 (1:2500, Millipore, Billerica, MA, 04-953), Nitrotyrosine (1:750, Millipore, Billerica, MA, Ab5411), HIF-1 alpha (1:500, Thermo Fisher, Rockford, IL, PA3-16521), pimonidazole (1:100, Hypoxyprobe, Burlington, MA, HP2-200), Heat Shock Factor HSF (1:100, Enzo, Farmingdale, NY, ADI-SPA-950).

Djamali A., Wilson N.A., Sadowski E.A., Zha W., Niles D., Hafez O., Dorn J.R., Mehner T.R., Grimm P.C., Hoffmann F.M., Zhong W., Fain S.B, & Reese S.R. (2016). Nox2 and Cyclosporine-Induced Renal Hypoxia. Transplantation, 100(6), 1198-1210.

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TGF-β Signaling Pathway Protein Analysis

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After treatment, the total proteins (25 μg) were separated via 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 10% skim milk for 1h at room temperature and incubated overnight at 4 °C with primary antibodies against phospho-Smad2 (1:1000, Millipore), Smad2 (1:200, Invitrogen), phospho-Smad3 (1:1000, Cell Signaling, Danvers, MA, USA), Smad3 (1:1000, Cell Signaling), TGF-β1 (1:1000, Santa Cruz, Dallas, TX, USA), and GAPDH (1:2000, Cell Signaling). After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (P0448, Dako, Glostup, Denmark) for 1 h. The membrane was detected using ECL reagents (Amersham Bioscience, Piscataway, NJ, USA) and visualized on an ImageQuant™ LAS 4000 system (GE Healthcare Life Sciences, Tokyo, Japan). The protein band densities were quantified using the Scion Image software (Scion, Frederick, MD, USA).

Lim J.H., Yook J.M., Oh S.H., Jeon S.J., Noh H.W., Jung H.Y., Choi J.Y., Cho J.H., Kim C.D., Kim Y.L, & Park S.H. (2021). Paricalcitol Improves Hypoxia-Induced and TGF-β1-Induced Injury in Kidney Pericytes. International Journal of Molecular Sciences, 22(18), 9751.

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Protein Expression Analysis by Western Blot

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Proteins were separated by SDS-PAGE, transferred to Immobilon-P (Millipore) and proteins were visualized using SuperSignal West Pico ECL (Pierce). Primary antibodies were against β-catenin (BD Transduction Labs 610153), phospho-Akt (Cell Signaling 9277), phospho-Smad2 (Millipore AB3849), Smad4 (Millipore 04-1033), Tgfbr2 (Novus NBP1-19434) and γ-tubulin (Sigma T6557). Blots were quantified by densitometry using Image J.

Bjerke G.A., Pietrzak K., Melhuish T.A., Frierson Jr. H.F., Paschal B.M, & Wotton D. (2014). Prostate Cancer Induced by Loss of Apc Is Restrained by TGFβ Signaling. PLoS ONE, 9(3), e92800.

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Western Blot Analysis of Lung Proteins

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Lung tissues and cells were homogenized in lysis buffer with protease inhibitor cocktail (Roche, IN, USA)^{25 (link)}. After determining the protein concentration by DC protein assay kits (Bio-Rad, Hercules, CA, USA), total soluble proteins (20 μg) were separated on 4–12% gradient gels (Invitrogen, CA, USA)^{25 (link)}. The proteins were transferred to a nitrocellulose membrane (Amersham, Little Chalfont, UK). Nonspecific binding was blocked with 5% skim milk (BD, CA, USA) at room temperature for 1 h^{25 (link)}. Then, the protein blots were probed with specific primary antibodies (1:1000 dilutions) against phospho-Smad1/5 (Cell Signaling, Frankfurt, Germany), phospho-Smad2 (Millipore, MA, USA), phospho-Smad3 (Millipore, MA, USA), PECAM-1 (Millipore, MA, USA), and L- and S-endoglin (R&D Systems, Minneapolis, MN, USA) overnight at 4 °C. After incubating with host-specific secondary antibodies (goat anti-rabbit IgG-HRP) for 1 h at room temperature, the immunoreaction was detected with a chemiluminescent substrate kit (Thermo Scientific, Rockford, IL, USA) and quantitated by densitometric scanning of the X-ray film with SLB MyImager (UVP Inc, Upland, CA, USA)^{25 (link)}. The blots were normalized for protein loading by washing in stripping solution and reprobing with a monoclonal antibody against β-actin (Cell Signaling, Frankfurt, Germany)^{25 (link)}.

Lee Y., Lee J., Nam S.K, & Hoon Jun Y. (2020). S-endoglin expression is induced in hyperoxia and contributes to altered pulmonary angiogenesis in bronchopulmonary dysplasia development. Scientific Reports, 10, 3043.

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Cytometric Analysis of Aortic Remodeling

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We performed imaging cytometric analysis using ArrayScan XTI (Thermo Fisher Scientific) for mouse aortae 1 week after the Ca + AngII treatment. Two aortic tissue sections were obtained from each mouse; WT (control; n = 4, Ca + AngII; n = 3) and TNC-KO (control; n = 5, Ca + AngII; n = 5). The tissue sections were stained for either NFκB (Cell Signaling Technologies), phospho-Stat3 (P-Tyr705, Cell Signaling Technologies), phospho-Smad2 (P-Ser465/467, Millipore) and CD45 (Abcam) antibodies with TSA labeling kit with AlexaFluor 488 tyramide (Invitrogen). All of the tissue sections were stained for smooth muscle α-actin (αSMA; Sigma-Aldrich) with DyLight 549-labeled secondary antibody (Jackson ImmunoResearch) and nuclei with DAPI. On average 339 cells/mouse were counted to obtain the cytometric data. For αSMA staining, the same staining and image acquisition protocols were used for all of the samples. For NFκB, phospho-Stat3, phospho-Smad2 and CD45, the staining protocols were optimized for individual target molecules, and a constant image acquisition protocol was used within the same target molecule. The cytometric data obtained by ArrayScan XTI were analyzed by FlowJo software. Immunohistochemical stainings were performed for type 1 collagen (LSL), fibronectin (Sigma-Aldrich) and lysyl oxidase (US Biological).

Kimura T., Shiraishi K., Furusho A., Ito S., Hirakata S., Nishida N., Yoshimura K., Imanaka-Yoshida K., Yoshida T., Ikeda Y., Miyamoto T., Ueno T., Hamano K., Hiroe M., Aonuma K., Matsuzaki M., Imaizumi T, & Aoki H. (2014). Tenascin C protects aorta from acute dissection in mice. Scientific Reports, 4, 4051.

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