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Anti phospho smad1 5

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Anti-phospho-SMAD1/5 is a lab equipment product that specifically recognizes and binds to the phosphorylated forms of SMAD1 and SMAD5 proteins. SMAD1 and SMAD5 are key components of the BMP signaling pathway.

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Lab products found in correlation

Anti smad1, by Cell Signaling Technology (3 mentions) Anti smad5, by Cell Signaling Technology (2 mentions) Anti phospho smad2, by Cell Signaling Technology (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Anti ctsk, by Santa Cruz Biotechnology (1 mentions) To pro 3, by Thermo Fisher Scientific (1 mentions) C2 confocal microscope, by Nikon (1 mentions) Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Nis elements microscope imaging software, by Nikon (1 mentions) Anti β galactosidase, by Promega (1 mentions) Can get signal immunostain solution b, by Toyobo (1 mentions) Tcs sp5 confocal microscope, by Leica camera (1 mentions) Vectashield, by Vector Laboratories (1 mentions) Anti sox2, by Cell Signaling Technology (1 mentions) Anti gfap, by BD (1 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions) Anti olig2, by Merck Group (1 mentions) Anti β actin peroxidase conjugated antibody, by Merck Group (1 mentions) Anti phospho akt, by Merck Group (1 mentions) Anti phospho egf receptor, by Cell Signaling Technology (1 mentions)

11 protocols using anti phospho smad1 5

1

Immunohistochemistry of SMAD1/5 and CTSK

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Immunohistochemistry staining of formalin-fixed and paraffin-embedded tissue specimens was carried out using a primary antibody and a peroxidase-conjugated secondary antibody with appropriate concentration. Visualization of proteins was performed with diaminobenzidine and hematoxylin was used as counterstaining. The primary antibodies were anti-phospho-SMAD1/5 (Cell Signaling Technology, cat. 9516; 1:200) and anti-CTSK (Santa Cruz, cat. 48353; 1:200).
Wang S., Li G.X., Tan C.C., He R., Kang L.J., Lu J.T., Li X.Q., Wang Q.S., Liu P.F., Zhai Q.L, & Feng Y.M. (2019). FOXF2 reprograms breast cancer cells into bone metastasis seeds. Nature Communications, 10, 2707.
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2

Immunohistochemical Analysis of Phospho-Smad 1/5

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Embryos were fixed and treated as described in [26 (link)]. The primary antibody was polyclonal anti-Phospho-Smad 1/5 (Cell Signalling; 1:10), the secondary was Alexa Fluor 488 Goat Anti-Rabbit IgG (Invitrogen; 1:500), and nuclear staining was done with TO-PRO3 (Molecular Probes; 10 μM). Fluorescently labeled embryos were mounted in DAKO or 3:1 glycerol/PBS. Confocal images were collected using the Confocal Microscope C2+ (Nikon) and processed using NIS-Elements Microscope Imaging Software (Nikon) and FIJI image analysis software [12 (link)].
Hodar C, & Cambiazo V. (2018). The dorsoventral patterning of Musca domestica embryos: insights into BMP/Dpp evolution from the base of the lower cyclorraphan flies. EvoDevo, 9, 13.
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3

Confocal Imaging of Drosophila Wing Discs

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Protocol was described previously (Harmansa et al., 2015 (link)). Each fly cross was set together with control and >10 wing imaginal discs from each genotype were processed in parallel. If the genotype could be distinguished, experimental and control samples were processed in the same tube. A representative wing disc was shown for all the experiments. Following primary antibodies were used; anti-Dpp (1:100; Matthew Gibson), anti-phospho-Smad1/5 (1:200; Cell Signaling, 9516S), anti-Brk (1:1000; Gines Morata), anti-Wg (1:120; DSHB, University of Iowa), anti-Ptc (1:40; DSHB, University of Iowa), anti-β-Galactosidase (1:1000; Promega Z378A), anti-mCherry (1:5000; Nigg lab, University of Basel). All the primary antibodies except anti-Dpp antibody were diluted in 5% normal goat serum (NGS) (Sigma) in PBT (0.03% Triton X-100/PBS). Anti-Dpp antibody was diluted in 5% NGS in Can Get Signal Immunostain Solution B (TOYOBO). All secondary antibodies from the AlexaFluor series were used at 1:500 dilutions. Wing discs were mounted in Vectashield (H-1000, Vector Laboratories). Images of wing discs were obtained using a Leica TCS SP5 confocal microscope (section thickness 1 μm).
Matsuda S, & Affolter M. (2017). Dpp from the anterior stripe of cells is crucial for the growth of the Drosophila wing disc. eLife, 6, e22319.
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4

Molecular Mechanisms of Glioblastoma Response to Temozolomide

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Reagent and antibody sources were as follows: AG1478 (Calbiochem/Merck, Darmstadt, Germany), BMP4 (R&D Systems, Minneapolis, MN, USA), DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), temozolomide (TMZ) and anti-β-Actin-peroxidase conjugated antibody (Sigma-Aldrich, Munich, Germany), anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), anti-BIM, anti-cleaved caspase 3, anti-cleaved caspase 7, anti-cleaved PARP (poly (ADP-ribose) polymerase-1), anti-EGF Receptor, anti-phospho-EGF receptor (Tyr1068), anti-FOXO3a, anti-phospho-FOXO3a (Thr32), anti-phospho-FOXO3a (Ser253), anti-phospho-FOXO3a (Ser318/321), anti-phospho-Rb (Ser807/811), anti-SMAD1, anti-SMAD3, anti-SMAD4, anti-SMAD5, anti-phospho-SMAD1/5 (Ser463/465), anti-phospho-SMAD3 (Ser423/425), anti-p27Kip1, anti-SOX2 (Cell Signaling Technology, Beverly MA, USA), anti-OLIG2, anti-β-Tubulin beta III isoform (Millipore, Temecula, CA, USA), anti-CYCLIN B1, p21CIP1 (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-GFAP (BD Pharmingen San Jose, CA), anti-NESTIN (R&D Systems, Minneapolis, MN, USA), and anti-CYCLIN D1 (ThermoFisher Scientific, Waltham, MA USA).
Ciechomska I.A., Gielniewski B., Wojtas B., Kaminska B, & Mieczkowski J. (2020). EGFR/FOXO3a/BIM signaling pathway determines chemosensitivity of BMP4-differentiated glioma stem cells to temozolomide. Experimental & Molecular Medicine, 52(8), 1326-1340.
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5

Quantifying Protein Levels in MEFs

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MEFs grown on six wells were harvested in lysis buffer containing 25 mM Tris-HCl, pH 7.5, 300 mM NaCl and 1% Triton with protease and phosphatase inhibitors. Tissues were minced by a Dounce homogenizer using 1-2 ml RIPA buffer. A total of 30 µg of protein were separated on 10% SDS-PAGE and transferred to PVDF membranes. After blocking of the membranes by 5% dry milk/TTBS, membranes were incubated in following primary antibody solutions: anti-Smurf1 (Novus, 1D7); anti-Smurf2 (Abcam, EP629Y3); anti-Smad1 (Cell Signaling, 9743); anti-Smad2 (Abcam, EP784Y); anti-Smad3 (Abcam, ab28379), anti-Smad5 (Abcam, EP619Y), anti-phospho-Smad1/5 (Cell Signaling, 41D10), anti-phospho-Smad2 (Cell Signaling, 138D4); anti-phospho-Smad3 (Rockland, 600-401-919), GAPDH (Santa Cruz, 0411), HSC70 (Santa Cruz, B-6). Protein detection was carried out using HRP-coupled species-specific secondary antibodies and ECL solution, exposed to Hyperfilm ECL.
Tang Y., Tang L.Y., Xu X., Li C., Deng C, & Zhang Y.E. (2018). Generation of Smurf2 Conditional Knockout Mice. International Journal of Biological Sciences, 14(5), 542-548.
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6

Analyzing Signaling Pathways in C2C12 and BRITER Cells

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Whole cell extracts were collected from C2C12 or BRITER cells in complete lysis buffer (20 mM Tris, 150 mM NaCl, 50 mM NaF, 1% NP40 substitute, HALT protease inhibitor cocktail (ThermoScientific). Proteins were resolved by electrophoresis on pre-cast 10% NuPage Bis-Tris gels (Invitrogen) and transferred to PVDF membranes (Bio-Rad). Membranes were blocked in 5% BSA-TBS-0.5% Tween-20 for 15 minutes, then incubated at 4° overnight with primary antibodies. Antibodies used were: rabbit anti-phospho-p38 MAPK (cat. no. 9211), anti-p38 MAPK XP (8690), anti-phospho-SMAD1/5 (9516), anti-SMAD1 XP (6944), anti-phospho-SMAD2/3 (8828), anti-SMAD2/3 XP (8685), anti-phospho-JNK (4668), anti-SAPK/JNK (9252), anti-phospho-Akt XP (4060), pan anti-Akt (4691), anti-phospho ERK p42/p44 (4377), anti-ERK p42/p44 (9102), anti-phospho-MKK3/6 (12280), anti-MKK3 (8535), anti-phospho-TAK1 (4531), anti-TAK1 (5206), anti-HA (3274), and anti-FLAG (14793); all from Cell Signaling); and mouse anti-p38α (cat. no. 33-1300), anti-p38β (33-8700; both ThermoFisher) and anti-β-actin (Sigma A1978). Proteins were visualized with HRP-conjugated secondary antibodies (Bio-Rad) with WestPico (ThermoFisher) or Immobilon (Millipore) chemiluminescence reagents. Images were obtained and analyzed for relative densitometric relationships on a LI-COR C-DiGit scanner using Image Studio software.
Cook B., Rafiq R., Lee H., Banks K.M., El-Debs M., Chiaravalli J., Glickman J.F., Das B.C., Chen S, & Evans T. (2019). Discovery of a small molecule promoting mouse and human osteoblast differentiation via activation of p38 MAPK-beta. Cell chemical biology, 26(7), 926-935.e6.
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7

Immunofluorescent Analysis of Hindlimb Tissues

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Hindlimb samples were dissected and fixed in 4% PFA at 4°C overnight. After fixation, samples were washed three times, 10 minutes each in PBS. The fixed hindlimb samples were then soaked in 30% sucrose solution for 2 hours and embedded in O.C.T. Cryo-section was performed at 12 μm thickness in the sagittal orientation. Immunofluorescent staining was performed as previously described (28 (link)). The primary antibodies used include anti-phospho-Smad1/5 (Cell Signaling Technologies, catalog# 13820, 1:50 dilution) and anti-Sox9 (Santa Cruz, catalog# sc-20095, 1:25 dilution). The secondary antibody was Alexa Fluor 568-conjugated goat anti-rabbit IgG (H+L) (Thermo Fisher, catalog# A11011, 1:500 dilution).
Liu H., Xu J, & Jiang R. (2019). Mkx-deficient Mice Exhibit Hedgehog Signaling Dependent Ectopic Ossification in the Achilles Tendons. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 34(3), 557-569.
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8

Analysis of Smad and VEGFR Signaling

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Cell lysates were electrophoresed on SDS-polyacrylamide gels and transferred onto nitrocellulose blotting membranes (GE Healthcare, Freiburg, Germany). Blots were probed with monoclonal anti-Smad1, anti-Smad2/3, anti-phospho-Smad1/5, or anti-phospho-Smad2/3 antibodies (Cell Signaling Technologies, Danvers, MA, USA), followed by a horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich). Signals were detected using a chemiluminescence detection kit (Amersham Biosciences, Buckinghamshire, UK) and a luminescent image analyzer (LAS-3000; Fujifilm, Tokyo, Japan).
To evaluate VEGF receptor phosphorylation, HUVEC extracts (400 μg of protein) were mixed with 4 μg of VEGFR1 or VEGFR2 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Protein G-agarose beads (Invitrogen/ThermoFisher Scientific), and incubated at 4°C overnight with gentle rotation. Immune complexes were precipitated by centrifugation and analyzed by western blotting using an anti-phospho-tyrosine antibody (Santa Cruz Biotechnology).
Oh M.K, & Kim I.S. (2019). Involvement of placental growth factor upregulated via TGF-β1-ALK1-Smad1/5 signaling in prohaptoglobin-induced angiogenesis. PLoS ONE, 14(4), e0216289.
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9

Immunohistochemical Analysis of Mandibular Bone

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Immunohistochemistry staining was performed on 4-μm-thick coronal sections prepared from 6 mandibles of KO or control mice using a DAB substrate kit (Vector Laboratories, Burlingame, CA, USA) following the manufacturer’s instruction. The primary antibodies used for immunohistochemistry are: anti-phospho-ERK (9101, 1:200, Cell Signaling, Danvers, MA, USA), anti-Lef1 (C12A5, 1:200, Cell signaling, Danver, MA, USA), anti-β-catenin (sc-7963, Santa Cruz), anti-Sox2 (ab97959, Abcam, Cambridge, MA, USA), and anti-phospho-SMAD1/5 (9516, Cell Signaling). Methyl green was used for counterstaining.
Wu J., Tian Y., Han L., Liu C., Sun T., Li L., Yu Y., Lamichhane B., D’Souza R.N., Millar S.E., Krumlauf R., Ornitz D.M., Feng J.Q., Klein O., Zhao H., Zhang F., Linhardt R.J, & Wang X. (2020). FAM20B-catalyzed glycosaminoglycans control murine tooth number by restricting FGFR2b signaling. BMC Biology, 18, 87.
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10

Protein Expression Analysis in Tissue Samples

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Tissue specimens were lysed in radioimmunoprecipitation assay buffer supplemented with 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, a protease inhibitor mixture, and phosphatase inhibitor cocktail-2 and cocktail-3 (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations were measured using the Bradford assay (Thermo Fisher Scientific, Carlsbad, CA, USA), and equal amounts of protein from each sample were subjected to immunoblot assay. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-hepcidin, anti-ferritin (R&D, Minneapolis, MN, USA), anti-superoxide dismutase (SOD) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-NOX-2, anti-NOX-4, anti-phospho-STAT-3, anti-STAT-3, anti-phospho-SMAD1/5, anti-SMAD1, anti-SMAD5, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Beverly, MA, USA). Each experiment was performed in triplicate.
Kim H.B., Jun J.H., Shim J.K., Oh J.E., Lee C, & Kwak Y.L. (2021). Changes in Hepcidin Levels in an Animal Model of Anemia of Chronic Inflammation: Mechanistic Insights Related to Iron Supplementation and Hepcidin Regulation. Oxidative Medicine and Cellular Longevity, 2021, 4357756.
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