The protein concentration was determined using BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amount of proteins from each sample was subject to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% polyacrylamide gel. Resolved proteins were transferred onto a polyvinylidenedifluoride (PVDF) membrane (Millipore, Billerica, MA, USA), and then blocked for 1 hour at room temperature using Tris Buffered Saline Tween (TBST) with 5% Bull Serum Albumin (BSA). After that, blotted proteins were incubated at 4°C overnight with a 1:1000 dilution of anti-mouse collagen antibody (Millipore), anti-Smad3 antibody (Cell Signaling Technology), and anti-p-Smad3 antibody (Cell Signaling Technology), as well as 1:500 dilution of anti-human collagen antibody (Millipore), respectively. Anti-GAPDH antibody (Cell Signaling Technology) was used as an internal control. After three washes with TBST for 30 minutes, the blotted proteins were incubated with the secondary antibody for 1 hour at room temperature, which was horse-radish peroxidase-conjugated anti-mouse, anti-rabbit or anti-goat IgG. Specific proteins were detected using an enhanced chemiluminescence system (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the intensity of bands was quantified using ImageQuantTL software (General Electric Company, Fairfield, CT, USA).
Anti smad3 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-Smad3 antibody is a laboratory reagent that specifically binds to the Smad3 protein. Smad3 is a signaling molecule involved in the transforming growth factor-beta (TGF-β) signaling pathway. This antibody can be used to detect and study the expression and localization of Smad3 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti p smad3 antibody, by Cell Signaling Technology (4 mentions)
Anti smad2 antibody, by Cell Signaling Technology (4 mentions)
Anti gapdh antibody, by Cell Signaling Technology (2 mentions)
Anti vimentin antibody, by Cell Signaling Technology (2 mentions)
Anti gapdh antibody, by Merck Group (2 mentions)
Anti n cadherin antibody, by Cell Signaling Technology (2 mentions)
Anti ptbp1 antibody, by Cell Signaling Technology (2 mentions)
Anti ptbp2 antibody, by Abcam (2 mentions)
Anti ptbp3 antibody, by Merck Group (2 mentions)
Anti hnrnpk antibody, by Abcam (2 mentions)
Anti hnrnpm antibody, by Merck Group (2 mentions)
Anti fubp3 antibody, by Abcam (2 mentions)
Anti cpsf2 antibody, by Abcam (2 mentions)
Anti tgfβ2 antibody, by Abcam (2 mentions)
Anti p smad2 antibody, by Cell Signaling Technology (2 mentions)
Anti smad5 antibody, by Abcam (2 mentions)
Anti id2 antibody, by Abcam (2 mentions)
Anti zeb1 antibody, by Abcam (2 mentions)
Anti snai antibody, by Abcam (2 mentions)
Goat anti mouse or anti rabbit secondary antibody, by Merck Group (2 mentions)
11 protocols using anti smad3 antibody
1
Western Blot Analysis of Collagen and Smad3
2
Western Blot Analysis of EMT Regulators
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The Western blotting analysis was performed using standard procedures. The following primary antibodies were used in the experiments: anti-PTBP1 antibody (Cell Signaling Technology, Beverly, MA, USA), anti-PTBP2 antibody (Abcam, Cambridge, UK), anti-PTBP3 antibody (Sigma-Aldrich, St. Louis, MO, USA), anti-HNRNPK antibody (Abcam), anti-HNRNPM antibody (Sigma-Aldrich), anti-FUBP3 antibody (Abcam), anti-CPSF2 antibody (Abcam), anti-G3BP2 antibody (Atlas Antibodies), anti-TGFβ1 antibody (Proteintech Group), anti-TGFβ2 antibody (Abcam), anti-p-SMAD2 antibody (Cell Signaling Technology), anti-SMAD2 antibody (Cell Signaling Technology), anti-p-SMAD3 antibody (Cell Signaling Technology), anti-SMAD3 antibody (Cell Signaling Technology), anti-SMAD5 antibody (Abcam), anti-ID2 antibody (Abcam), anti-ZEB1 antibody (Abcam), anti-SNAI antibody (Abcam), anti-E-cadherin antibody (Proteintech Group), anti-Vimentin antibody (Cell Signaling Technology), anti-N-cadherin antibody (Cell Signaling Technology) and anti-GAPDH antibody (Sigma-Aldrich). The blots were incubated with a goat anti-rabbit or anti-mouse secondary antibody (Sigma-Aldrich) and visualized with a commercial ECL kit (Pierce, Rockford, IL).
3
Protein Expression Analysis in HK-2 Cells
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Proteins were obtained from HK-2 cell lysates and animal tissue, using RIPA buffer (Thermo, USA) containing Complete, Mini Protease Inhibitor Cocktail (Roche, USA) and Halt Phosphatase Inhibitor Cocktail (Thermo, USA). To examine the expression of proteins, separated protein in SDS/PAGE (gradient gels) were transferred onto polyvinylidene difluoride membranes (PVDF) (Bio-rad, USA), and then incubated with 5% BSA (sigma) for blocking. The membranes were incubated with anti-GAPDH antibody (Santacruz, USA), anti-type 1 collagen antibody (abcam, UK), anti-fibronectin antibody (abcam), anti-α-SMA antibody, anti-Vimentin antibody, anti-Lin28a antibody, anti-phospho-ERK antibody, anti-ERK antibody, anti-phospho-JNK antibody, anti-JNK antibody, anti-phospho-p38 antibody, anti-p38 antibody, anti-phospho-smad3 antibody and anti-smad3 antibody (Cell signaling, USA). After incubating with HRP-linked antibody (Cell signaling), the protein expression on blots was detected by ChemiDocTMXRS+ (Bio-rad) and the bands were quantified using the Image LabTM Software (Bio-rad).
4
Western Blot Analysis of TGF-β Signaling
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4T1 cells or tumor samples were lysed in TNEN buffer containing proteinase inhibitor cocktail. Proteins were then denatured at 95 °C and loaded onto 12% SDS-PAGE gel, and transferred to PVDF membrane. After incubation with anti-β-Actin antibody (nb100-74340, Novus), anti-TGF-β1 antibody (Abcam, USA), anti-TGFβR1 antibody, anti-Smad2 antibody, anti-Smad3 antibody, or anti-Smad4 antibody (Cell Signaling, USA) at 4 °C overnight, the blots were incubated with HRP-conjugated secondary antibody (1:5000). Signals were visualized using ECL substrates (PerkinElmer, USA) and recorded by UVP BioSpectrum imaging system. The semi-quantitative analyses of the protein levels were performed by Launch VisionWorks LS. β-Actin was used as an internal control for normalization purpose.
5
Crosstalk between Notch and TGFβ Signaling
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Human urethral scar fibroblasts were starved in serum‐free medium for 4 h and then treated with TGFβ1 (10 ng/ml) for 1 h. Triton X‐100 cell lysates were immunoprecipitated with anti‐NICD antibodies that were covalently coupled to Sepharose beads (Amersham Biosciences). The beads were then incubated at 4°C for 2 h. Immunoblots were probed with anti‐Smad3 antibody (Cell Signaling).
6
Chromatin Immunoprecipitation of SMAD3
7
Western Blotting Analysis of Smad Signaling
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Sample collection and western blotting were performed as previously described [8 (link)]. The following primary antibodies were used: rabbit polyclonal anti-phosphorylated Smad2 antibody (#3108; Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-Smad2 antibody (#3103; Cell Signaling Technology), anti-phosphorylated Smad3 antibody (#9520; Cell Signaling Technology), anti-Smad3 antibody (#9523; Cell Signaling Technology), mouse monoclonal anti-α-SMA antibody (A2547; Sigma-Aldrich), and mouse monoclonal anti-α-tubulin antibody (T9026; Sigma). The intensity of each band was quantified using ImageJ software (version 1.48p; National Institutes of Health, Bethesda, MD, USA).
8
Quantitative Analysis of Carotid Artery Proteins
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Rat carotid arteries were pretreated with RIPA lysis buffer and protease inhibitors (Beyotime Biotechnology, Shanghai, China). Total proteins were determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific), separated by 10% SDS-PAGE gel electrophoresis, and transferred to PVDF membranes. The PVDF membranes were blocked with BSA (Sigma-Aldrich) and then hybridized with antibodies. Anti-FMOD antibody (catalog # ab267465) was purchased from Abcam (Cambridge, UK) and diluted 1:1000. Anti-Smad3 antibody (catalog # #9513) was purchased from Cell Signaling Technology (MA, USA) and diluted 1:1000. GAPDH was used as an internal reference and anti-GAPDH antibody was purchased from Abcam (catalog # ab9485) and diluted 1:1000. The PVDF membranes were incubated with a developing solution and developed in the Molecular Imager Gel Doc XR system (Bio-Rad Laboratories, Inc.). Western blots were analyzed with ImageJ software (National Institutes of Health).
9
Western Blot Analysis of Tumor Markers
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Total protein was extracted from tumor samples or cells. The protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking via 5% skimmed milk, membranes were incubated (overnight, 4°C) with specific primary antibodies as follows: anti-HOXB7 antibody, anti-NANOG antibody, anti-EPCAM antibody, anti-ERK antibody, anti-phosphorylated ERK antibody, anti-c-Myc antibody, anti-E-cadherin antibody, anti-N-cadherin antibody, anti-Slug antibody, anti-Vimentin antibody, anti-α-SMA antibody (Abcam); anti-MMP2 antibody, anti-MMP9 antibody, anti-GAPDH antibody, anti-SMAD3 antibody, anti-phosphorylated SMAD3, anti-p38 antibody, anti-phosphorylated p38, anti-AKT antibody, anti-phosphorylated AKT antibody (Cell Signaling Technology, Danvers, MA, USA). After washing, membranes were incubated 2 h with secondary antibodies (Sigma-Aldrich). The protein intensity was determined and measured by Image Lab software (5.2.1 Version, Bio-Rad Laboratories Co. Ltd, CA, USA). After normalization to GAPDH protein units for each sample, the semi-quantitate results for either tumor or adjacent samples were obtained as a ratio.
10
Western Blot Analysis of Smad Signaling
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Cells were homogenized in RIPA buffer containing 1% protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). The protein levels were quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Amounts of 40 μg of total cell lysates were loaded and separated with 10% SDS-PAGE, and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk in TBST buffer at room temperature for 1 h, followed by incubation at 4 °C overnight with the primary antibodies, including an anti-p-Smad2 antibody, anti-p-Smad3 antibody, anti-Smad2 antibody, anti-Smad3 antibody (Cell Signaling, Danvers, MA, USA), and anti-β-actin antibody (Thermo Fisher Scientific, Waltham, MA, USA). Then, the membranes were incubated with horseradish-peroxidase-conjugated IgG secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. The signals were detected using a chemiluminescence detection kit (Millipore, Burlington, MA, USA). The intensity of the protein was quantified using the ImageJ software.
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