The anti-human B7-H3, MMP2 and MMP9 antibodies used for immunohistochemistry were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, United States). The anti-human B7-H3, E-Cadherin, N-Cadherin, Vimentin, β-catenin, CD44, Oct4, Akt, phosphorylated Akt, Smad1 and Smad9 antibodies were from Abcam (Cambridge, MA, United States). The anti-human phosphorylated Smad1/5 and Smad1/5/9 antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, United States). The anti-human CD133 antibody for flow cytometry was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The horseradish peroxidase-conjugated secondary anti-mouse and anti-rabbit antibodies and the GAPDH antibody were from Beyotime (Nantong, China). The fluorescent dye DiI that was used to label the cell membrane was from Keygentec (Nanjing, China). The Jak2-specific inhibitor AG490 was purchased from Sigma-Aldrich (St. Louis, MO, United States). The PI3K-specific inhibitor LY294002 was from Selleckchem (Houston, TX, United States). The MatrigelTM matrix used in the invasion assay was from BD (San Jose, CA, United States).
Smad1
Manufactured by Abcam
Sourced in United Kingdom, United States
Smad1 is a protein involved in the transforming growth factor-beta (TGF-β) signaling pathway. It acts as an intracellular signal transducer and transcriptional modulator, mediating the signal from the cell surface to the nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Abcam (3 mentions)
P smad1, by Abcam (2 mentions)
Tgf β1, by Abcam (2 mentions)
Smad5, by Abcam (2 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Mmp 2, by Santa Cruz Biotechnology (1 mentions)
Anti human b7 h3, by Abcam (1 mentions)
Smad1 5 9, by Cell Signaling Technology (1 mentions)
Ag490, by Merck Group (1 mentions)
Matrigeltm matrix, by BD (1 mentions)
Smad9, by Abcam (1 mentions)
Ly294002, by Selleck Chemicals (1 mentions)
Vimentin, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
Gapdh antibody, by Beyotime (1 mentions)
Nunc lab tec 2 16 well glass chamber slides, by Thermo Fisher Scientific (1 mentions)
Bmprii, by BD (1 mentions)
Duolink in situ proximity ligation, by Merck Group (1 mentions)
9 protocols using smad1
1
Analyzing Tumor Stem Cell Markers
2
In Situ Proximity Ligation for Protein Interactions
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Immortalised human myoblasts seeded in Nunc Lab-Tec II 16-well glass chamber slides (Thermo Scientific) were subjected to Duolink in situ proximity ligation (Sigma-Aldrich) as previously described77 (link) using the following antibodies: BMPRII (#612292, BD Biosciences), IRS4 EP907Y (#TA303856, Origene), Smad1 (ab55476, Abcam), Smad4 (#9515, Cell Signaling).
3
Protein Extraction and Western Blot Analysis
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Total protein was extracted from the cultivated cells with radioimmunoprecipitation assay solution (Thermo Fisher, Beijing, China). After denaturing, proteins samples were subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE was then transferred to polyvinylidene fluoride (PVDF) membranes, followed by blocking for 2 hours at 25 °C in 5% non-fat milk. Subsequently, The PVDF membranes were sealed with 5% skimmed milk at 37 °C for 120 min and then, overnight incubated at 4 °C with the following primary antibodies: SOX2, NANOG, ALDH1A1, TGF-β1, TGFβ-R1, SMAD1, P-SMAD1, SMAD2, and P-SMAD2 (all Abcam, Beijing, China). Subsequently, PVDF membranes were incubated for 60 min at 37 °C with goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721, 1:1,000, Abcam, Cambridge, MA, USA). After washing, signals were visualized using a ChemiDoc XRS imaging system and Quantity One analysis software (Bio-Rad, San Francisco, California, USA).
4
Protein Expression Quantification Protocol
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Proteins were obtained by radioimmunoprecipitation assay lysis buffer (Abcam Corp, USA) and quantified by bicinchoninic acid. Protein, 50 μg per sample, was separated onto 10% SDS–PAGE gels and transferred onto a polyvinyl difluoride (PVDF) filter membrane. Then, PVDF membranes were incubated with 5% non-fat dried milk, anti-RRBP1 (Abcam Corp., USA), Smad-1 (Abcam Corp., USA), p-Smad-1 (Abcam Corp., USA), Smad-3 (Abcam Corp., USA), p-Smad-3 (Abcam Corp., USA), TGF-β1 (Abcam Corp., USA) and anti-GAPDH (Abcam Corp., USA), respectively. Finally, membranes were washed with phosphate-buffered saline (PBS) and incubated with secondary antibodies. Signals were developed using an enhanced chemiluminescence reaction kit (Applygen Technologies, Beijing, China).
5
Western Blot Analysis of Signaling Pathways
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EB cell lysates were prepared using 1 × RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% Triton, 1% sodium deoxycholate and 0.1% SDS) supplemented with Complete Protease Inhibitor Cocktail (Roche) and PhosSTOP (Roche), and quantified with Bradford reagent (Sigma). Samples were prepared in Laemmli buffer (BioRad) and loaded on gels for SDS–polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinyl difluoride membrane (Millipore). The following primary antibodies were applied at the indicated dilution in Primary Antibody Signal Boost Immunoreaction Enhancer (Calbiochem); phosphorylated Smad1/5/8 (S465/8 and S426/8, 1:1,000, Cell Signaling), Smad1 (1:3,000, Abcam), cTnI (1:1,000, Abcam), β-active Catenin (1:1,000, Millipore), pGsk3α/β (S21/S9, 1:1,000, Cell Signaling). Gapdh (Abcam) and actin (Millipore) were diluted at 1:3,000 with 5% BSA in 1 × TBS–Tween 20. All primary antibodies were incubated for 12 h at 4 °C on a shaker. Enhanced chemiluminescence peroxidase-labelled anti-mouse and anti-rabbit antibodies (GE Biosciences) were diluted at 1:20,000 with 5% BSA in 1 × TBS–Tween 20. After washing the membranes, SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific) was utilized to detect the horseradish peroxidase signal. All uncropped western blots can be found in Supplementary Fig. 4 .
6
Western Blot for Bone Marker Proteins
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For the western blot analysis, each protein extract (30 μg of total protein) was resolved by SDS-PAGE using 10% polyacrylamide gels and subsequently transferred to Immun-Blot® PVDF membranes (Bio-Rad, Hercules, CA, USA). The primary antibodies and their dilution factors were as follows BST2, 1:1000 (Santa Cruz Biotechnology, Dallas, TX, USA); RUNX2, 1:1000 (Santa Cruz Biotechnology); SMAD1, 1:1000 (Abcam, Cambridge, UK); SMAD4, 1:500 (Abcam); SMAD5. 1:500 (Abcam); SMAD 8, 1:500 (Abcam); T-SMAD1/5/8, 1:500 (Santa Cruz Biotechnology); p-SMAD1/5/8, 1:500 (Santa Cruz Biotechnology), and β-actin, 1:1000 (Sigma-Aldrich). Secondary antibodies were used at a 1:5000 dilution. The detection of protein bands was facilitated by an Enhanced Chemiluminescence Kit (Elpis Biotech) and membranes exposures to X-ray film (Amersham, Buckinghamshire, UK).
7
Cartilage Protein Extraction and Analysis
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Total protein of cartilage tissues was extracted by tissue total protein extraction kit (Beyotime, China). SDS-PAGE was performed (90 V-30 min, 120 V-60 min), and the proteins were transferred to a wet PVDF membrane (400 mA-90 min), which was blocked with 5% no-fat milk at 37°C for 2 h, and incubated with primary antibodies of Smad1, TGF-β1 MMP-1, MMP-3, MMP-13, or GAPDH (1: 1000, Abcam, UK) at 4°C overnight. Then, the primary antibodies were removed, and the membrane was washed 3 times with TBST before incubating with goat anti-rabbit secondary antibodies (1: 3000, Abcam, UK) at 37°C for 2 h. The membrane was washed 3 times with TBST before image development. The band density of target proteins was normalized against GAPDH levels to be presented as the final results.
8
Immunohistochemical Analysis of Tumor Tissues
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The tumour tissues were xed in 4% paraformaldehyde for 24 hours, followed by dehydrating in a graded alcohol series and embedding in para n, followed by cutting into 5μm sections. The sections were depara nised, rehydrated with a graded alcohol series and then incubated in 96℃ with 0.01 mol/l sodium citrate buffer for the antigen retrieval. Following the incubation in 5% H 2 O 2 for a period of 2 hours, the sections were incubated using primary antibodies including ki67 and SMAD1 (Abcam, England) overnight at 4℃. Immunostaining was carried out with the use of streptavidin-peroxidase and diaminobenzidinef (DAB) following the manufacturer's instructions (Beyotime, Shanghai, China). Eventually, the sections were not only observed under a uorescence microscope (Leica, Wetzlar, Germany) but also imaged.
9
Protein Expression Analysis of hBMMSCs
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The hBMMSCs were cultured in 60-mm dishes and incubated with 1× radioimmunoprecipitation assay lysis buffer (Huaxingbio) containing a mixture of PMSF (Huaxingbio) and protease inhibitor (Huaxingbio). After centrifugation on ice, the protein concentration in the supernatant was measured by the bicinchoninic acid approach. The same amount of protein was split on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a PVDF membrane (Millipore). The membranes were incubated in rapid blocking solution to block nonspeci c binding, then incubated with primary antibodies against BMP2, RUNX2, OSX, SMAD1, SMAD5, SMAD1/5 (Abcam), pSMAD1/5 (Cell Signaling Technology), or GAPDH (Abcam). Then, the membranes were incubated with the secondary antibody for 1 h. Finally, the immunoreacted protein bands were detected by enhanced chemiluminescence.
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