For phosphorylated SMAD1/5/8 Western blotting, lysates from cells that were serum-deprived in DMEM containing 0.1% BSA for 5 to 6 hours and treated with BMP4 for 15 min were prepared in extraction buffer containing 10 mM tris-HCl (pH 7.4), 1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium fluoride, and 1× EDTA-free protease inhibitor cocktail (Roche cOfmplete). Lysates were cleared by centrifugation at 13,000 rpm for 10 min at 4°C. Total protein was measured by BCA (Pierce). Equal amounts of protein were separated on 5 to 15% gradient SDS–polyacrylamide gel electrophoresis gels and immunoblotted with phosphorylated SMAD1/5/8 antibody (Cell Signaling Technology). Antiphosphotyrosine antibody (4G10) was used for immunoprecipitations of phosphorylated MuSK (Millipore). Membranes were then stripped and reprobed with SMAD1/5/8 antibody (Cell Signaling Technology) or MuSK antibody for phospho-MuSK immunoprecipitation (R&D Systems).
Smad1 5 8 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The SMAD1/5/8 antibody is a laboratory research tool used to detect the presence and levels of the SMAD1, SMAD5, and SMAD8 proteins in biological samples. These proteins are important mediators in the transforming growth factor-beta (TGF-β) signaling pathway, which regulates various cellular processes. The antibody can be used in techniques such as Western blotting, immunohistochemistry, and immunofluorescence to identify and quantify these proteins in experimental settings.
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2 protocols using smad1 5 8 antibody
1
Analyzing Phosphorylated SMAD1/5/8 in BMP4-Treated Cells
2
Western Blot Analysis of ABCA1, ABCG1, SR-A, CD36, and BMPR-2
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RAW 246.7 cells were lysed with 200 μL lysis buffer containing 20 mmol/L HEPES, 25 mmol/L MgCl, 5 mmol/L KCL, 0.5% (v/v) complete protease inhibitor, and Triton X-100. Then, the debris was removed by centrifugation at 12,000× g at 4 °C for 10 min. Equal amounts of cell protein (typically 80 μg) were separated using 8% precast SDS-PAGE gels (Invitrogen) and electrophoretically transferred to PVDF membrane. The membranes were subsequently probed individually with 1:150 polyclonal primary ABCA1 antibody, ABCG1 antibody, SR-A antibody, CD36 antibody, or BMPR-2 antibody (BD Transduction Laboratories, San Jose, CA, USA) or 1:1000 SMAD1/5/8 antibody (Cell Signaling Technology, Beverly, MA, USA). Detection was by incubation with goat anti-mouse immunoglobulin G (IgG; 1:5000; Sigma) followed by enhanced chemiluminescence (ECL, Amersham Pharmacia, NJ, USA). The intensity of the bands was measured using labwords analysis software (Shenteng, Shanghai, China).
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