Dulbecco’s modified Eagle’s medium GlutaMAX (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, protease and phosphatase inhibitors cocktail were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant human sRANKL (Invitrogen_PHP0034) was purchased from Thermo Fisher Scientific. Anti-NFATc1 (sc-7294) and anti-c-Fos (sc-166940) polyclonal antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-phospho-Smad1/5/9 and anti-GAPDH antibodies were obtained from Cell Signaling (Cell Signaling Technology, Danvers, MA, USA). Primers for RT-PCR (QuantiTect primer assay, Table S1 ) and RNA extraction kit (RNeasy kit) were purchased from Qiagen (Hilden, Germany). The primers for Cathepsin K and c-Fos were designed as already described [10 (link)] and ordered from Microsynth (Balgach, Switzerland). TRAP staining solution (leukocyte acid phosphatase kit, 386-A) was purchased from Sigma. All others chemicals were obtained from Sigma (St. Louis, MO, USA).
Anti phospho smad1 5 9
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-Smad1/5/9 is a primary antibody that specifically recognizes the phosphorylated forms of Smad1, Smad5, and Smad9 proteins. Smad proteins are key intracellular mediators of TGF-beta and BMP signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (2 mentions)
Supersignal west pico chemiluminescence substrate, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Anti tsp 1, by Thermo Fisher Scientific (2 mentions)
Cyclohexamide, by Merck Group (2 mentions)
Sb431542, by Cayman Chemical (2 mentions)
Anti smad1, by Cell Signaling Technology (2 mentions)
Human tgf β1, by R&D Systems (2 mentions)
Ripa biffer, by Thermo Fisher Scientific (2 mentions)
Anti tsp 4, by R&D Systems (2 mentions)
Anti phospho smad2, by Cell Signaling Technology (2 mentions)
Anti phospho smad3, by Cell Signaling Technology (2 mentions)
Phosphatase inhibitors cocktail, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Anti nfatc1 sc 7294, by Santa Cruz Biotechnology (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase labeled secondary antibody, by GE Healthcare (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
5 protocols using anti phospho smad1 5 9
1
Osteoclast Differentiation Assay
2
Immunohistochemistry and In Situ Hybridization
3
TGF-β1 Signaling in Confluent EC
4
Comprehensive Antibody Characterization Protocol
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Primary antibodies used were as follows: Anti-CD133 (W6B3C1) [1:10 for immunofluorescence (IF), 1:100 for immunoblotting (WB), 130-092-395, Miltenyi Biotec], anti-Smad1 (1:1,000 for WB, cat. no. 9743; Cell Signaling Technology), anti-phospho-Smad1/5/9 (1:1,000 for WB, cat. no. 13820; Cell Signaling Technology), anti-Smad4 (1:1,000 for WB, 1:500 for IF, cat. no. 46535; Cell Signaling Technology), anti-PCNA (1:1,000 for WB, NB100-456; Novus Biologicals), anti-tubulin (1:1,000 for WB, T5326; Sigma-Aldrich), anti-GAPDH (1:1,000 for WB, 60004-1-Ig; Proteintech), and anti-MKLP-1 (1:100 for IF, sc-867; Santa Cruz Biotechnology). Horseradish peroxidase-labeled secondary antibodies were purchased from the General Electric (GE) Healthcare and used at 1:10,000. Fluorescence-labeled Alexa secondary antibodies used in this study were obtained from Molecular Probes and used at 1:500.
5
TGF-β1 Signaling in Confluent EC
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Confluent EC were treated with DMSO or human TGF-β1 (R&D, 10ng/ml) for 24 hours. Cells were washed twice with ice-cold PBS and lysed using RIPA Biffer (Thermo Scientific, 25mM Tris•HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS).
30 μg of protein was separated using 8% SDS-PAGE and transferred to PVDF membrane (Bio-Rad). Membranes were incubated with the anti-TSP-4 (R&D); anti-TSP-1 (Thermo Fisher); anti-phospho-SMAD3, anti-phospho-SMAD2, anti-phospho-SMAD1/5/9, and anti-SMAD1 (Cell Signaling); anti-β-actin (Sigma) primary antibodies followed by horseradish peroxidase–linked IgG; and visualized by chemiluminescence using Super Signal West Pico Chemiluminescence substrate (Thermo Fisher).
Cells were pre-treated with 2.5 μM SIS3 (Calbiochem), 10 μM SB431542 (Cayman Chemical) and cyclohexamide (Sigma-Aldrich, 10 μg/ml) for 30 min before TGF-β1 stimulation.
30 μg of protein was separated using 8% SDS-PAGE and transferred to PVDF membrane (Bio-Rad). Membranes were incubated with the anti-TSP-4 (R&D); anti-TSP-1 (Thermo Fisher); anti-phospho-SMAD3, anti-phospho-SMAD2, anti-phospho-SMAD1/5/9, and anti-SMAD1 (Cell Signaling); anti-β-actin (Sigma) primary antibodies followed by horseradish peroxidase–linked IgG; and visualized by chemiluminescence using Super Signal West Pico Chemiluminescence substrate (Thermo Fisher).
Cells were pre-treated with 2.5 μM SIS3 (Calbiochem), 10 μM SB431542 (Cayman Chemical) and cyclohexamide (Sigma-Aldrich, 10 μg/ml) for 30 min before TGF-β1 stimulation.
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