RIPA lysis buffer (Beyotime, Beijing, China) was used to extract total protein from cells, and a BCA protein assay kit (Beyotime) was used to measure the protein concentrations following the manufacturer’s protocols. The protein samples were separated and transferred using 12% SDS-PAGE and PVDF membranes. The membranes were blocked in skimmed milk (5%) at room temperature for 2 h. After that, the membranes were cultured with primary antibodies at 4 °C overnight. Primary antibodies includes anti-Smad3 antibody (1:1000, Abcam), anti-ALP antibody (1:1000, Abcam), anti-Osteopontin (OPN) antibody (1:1000, Abcam), anti-Runx-2 antibody (1:1000, Abcam), anti-Osterix antibody (1:1000, Abcam), and anti-GAPDH antibodies (1:2000, Abcam). In the next day, the membranes were incubated with a horseradish peroxidase-conjugate secondary antibody (1:2000, Santa Cruz) for 1 h at room temperature. The enhanced chemiluminescence detection system (ECL, Roche Molecular Biochemicals) was used to measure the blots.
Anti smad3 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The Anti-Smad3 antibody is a laboratory reagent used in research applications. It is designed to detect and bind to the Smad3 protein, which is a key mediator in the transforming growth factor-beta (TGF-β) signaling pathway. This antibody can be used to study the role of Smad3 in various cellular processes and disease states.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Anti osterix antibody, by Abcam (1 mentions)
Horseradish peroxidase conjugate secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti alp antibody, by Abcam (1 mentions)
Anti runx2 antibody, by Abcam (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ecl western blotting detection reagent, by Merck Group (1 mentions)
Goat anti rabbit igg conjugated to horseradish peroxidase, by Abcam (1 mentions)
Mouse monoclonal β actin antibody, by Merck Group (1 mentions)
Normal rabbit igg, by Cell Signaling Technology (1 mentions)
Ez chip, by Merck Group (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Sangon (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
6 protocols using anti smad3 antibody
1
Protein Expression Analysis in Cells
2
Smad3 Protein Expression Analysis
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SW1353 cells were lysed using RIPA buffer with protease inhibitor cocktail (Millipore, U.S.A.). The protein concentration of cell lysates was quantified by BCA Kit, and 50 ng of protein was separated by SDS/PAGE (8% gel) and then transferred onto a PVDF membrane (Millipore, U.S.A.). The membranes were blocked in 5% non-fat dry milk diluted with TBST at room temperature (RT) for 1 h and probed overnight at 4°C with primary antibody, as follow: anti-Smad3 antibody (Abcam, U.S.A.). After that, the membranes were washed by TBST and incubated with a goat anti-rabbit IgG conjugated to horseradish peroxidase (Abcam, U.S.A.) for 1 h at RT. Incubation with monoclonal mouse β-actin antibody (1:10000 dilution; Sigma, U.S.A.) was performed as the loading control. The proteins were visualized using ECL Western blotting detection reagents (Millipore, U.S.A.). The densitometry of the bands was quantified using the ImageJ 1.38X software (U.S.A.).
3
SMAD3 Chromatin Immunoprecipitation Assay
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ChIP assays were performed essentially as described before[14 (link)] with anti-SMAD3 antibody (Abcam). Precipitated genomic DNA was amplified by real-time PCR with primers as previously described[3 (link),11 (link),15 (link)].
4
ChIP Analysis of Smad3 Transcription Factor
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ChIP analysis was performed using EZ-ChIP (Millipore), Dynabeads Protein G (Thermo Fisher Scientific) and anti-Smad3 antibody (Abcam) or normal rabbit IgG (Cell Signaling Technology) as a negative control. Information on the antibodies used may be found in Supplementary Data 4 . Immunoprecipitated DNA was quantified by qRT-PCR. For information on primers used, see Supplementary Data 3 .
5
Immunofluorescence Staining of Smad3
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2 × 104 cells/well were seeded on glass slides coated with 0.1% Gelatin (Gibco) in a 24-well plate (5 × 104 cells/well) and cultured in a different medium for 48 h before staining. Then, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with the blocking buffer (1% bovine serum albumin (Sangon Biotech) and 0.02% Triton X-100 (Sigma) in phosphate-buffered saline), and stained with antibodies according to the manufacturer's instructions. The anti-Smad3 antibody was obtained from Abcam. DAPI was obtained from Sangon Biotech. Goat anti-rabbit Alexa Fluor 488 was from Invitrogen.
6
Western Blot Analysis of BMPR-II Pathway
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ASMC protein was isolated using RIPA buffer (Sangon biotech, Shanghai, China), and the protein concentration was determined using the BCA method (Yeasen Biotech, Shanghai, China) as previous paper described [19 (link)]. Thirty micrograms of protein per well were run on 10% SDS-polyacrylamide (sodium dodecyl sulphate polyacrylamide) gel electrophoresis and then transferred to PVDF membranes (Millipore, USA). Blots were blocked in 5% bovine serum albumin at room temperature for 1 h and incubated with primary antibodies overnight at 4 ℃ followed by incubation with corresponding secondary antibodies at room temperature for 1 h. Then, membranes were exposed to ECL reagent (Junxin, Suzhou, China). β-actin was measured as a loading control. Antibodies used in this study are listed below: anti-BMPR-II antibody (Abcam 130206), anti-SMAD3 antibody (Abcam 208182), anti-pSMAD3 antibody (Abcam 118825), anti-MRTF antibody (Abcam 49311) and anti-β-actin antibody (Abcam 8226) was obtained from Abcam (Cambridge, UK) and operated according to the manufacturer’s instructions.
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