Total protein was extracted from embryonic brain tissues and cultured mNSCs. The protein concentrations were determined using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the lysates were then mixed fully with a 5× sample buffer. Equal protein amounts (30 µg) were subjected to SDS-PAGE and electrotransferred onto a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The protein expression of p-Smad1/5/8 (Cell Signaling Technology, Boston, MA, USA, dilution rate, 1:1000) was detected with Western blotting. The membrane was stripped for an hour after the p-Smad1/5/8 had been photographed, and then blocked to detect the loading control (β-actin, Cell Signaling Technology, Boston, MA, USA, dilution rate, 1:1000), washed with TBST for three times, and imaged with a gel imaging system (Tanon Science & Technology, Shanghai, China). The individual band in the Western blot was semiquantified with ImageJ software.
P smad1 5 8
Manufactured by Cell Signaling Technology
Sourced in United States
P-Smad1/5/8 is a laboratory reagent used to detect phosphorylated Smad1, Smad5, and Smad8 proteins. These Smad proteins are key mediators of the BMP signaling pathway. This product can be used in techniques such as Western blotting to analyze the activation status of the BMP signaling cascade.
Automatically generated - may contain errors
Lab products found in correlation
P erk1 2, by Cell Signaling Technology (7 mentions)
β actin, by Santa Cruz Biotechnology (7 mentions)
Smad1 5 8, by Santa Cruz Biotechnology (7 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
β catenin, by Cell Signaling Technology (6 mentions)
P jnk, by Cell Signaling Technology (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
β actin, by Merck Group (4 mentions)
P smad2, by Cell Signaling Technology (4 mentions)
Erk1 2, by Cell Signaling Technology (4 mentions)
P erk, by Cell Signaling Technology (4 mentions)
P gsk3β, by Cell Signaling Technology (4 mentions)
Gsk3β, by Cell Signaling Technology (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
P akt, by Cell Signaling Technology (4 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Runx2, by Cell Signaling Technology (3 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (2 mentions)
63 protocols using p smad1 5 8
1
Quantifying Smad1/5/8 Phosphorylation
2
Recombinant Human BMP-9 Production
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MB109 is a recombinantly expressed human BMP-9 cytokine. It contains a methionine residue in front of the mature domain of human BMP-9 (Ser320-Arg429, 110 residues). MB109 was prepared as described previously [13 ]. Recombinant BMP-2 was purchased from joint Protein Central (jointproteincentral.com , Incheon, Korea). The antibodies against p21, P-SMAD1/5/8 and P-p38 were purchased from Cell Signaling Technology (Massachusetts, USA), ID3 from Santa Cruz Biotechnology (California, USA), β-actin from Sigma Aldrich (Missouri, USA), CD90 from Novus Biologicals (CO, USA), AFP from Abcam (Cambridge, UK), PE-conjugated CD90 from BD Biosciences (New Jersey, USA) and FITC-conjugated CD44 from Miltenyi Biotec (Bergisch Gladbach, Germany). Chemical inhibitors, LDN193189 was purchased from Sellekchem (Texas, USA) and SB202190 was purchased from Sigma Aldrich (Missouri, USA). The cell culture media, fetal bovine serum (FBS), Penicillin-Streptomycin Solution (P/S) and Trypsin-EDTA were purchased from Hyclone (Utah, USA).
3
Immunoblotting and Immunohistochemistry of Smad Proteins
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2Tissues were homogenized in T-Per (Pierce Endogen, Rockford, IL) and quantitated with a bicinchoninic acid (BCA) protein assay kit. Sixty micrograms of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and probed with antibodies to the following targets: p-Smad 1/5/8 (Cell Signaling, Beverly, MA), t-Smad 1/5/8 (Santa Cruz Biotechnology, Paso Robles, CA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling). Densitometry was done with the VersaDoc Imaging System (Bio-Rad, Hercules, CA) and quantified using Image Lab (Bio-Rad). For immunohistochemistry, 10 micron sections of muscle were fixed in Bouin’s fixative for 15 min and then oxidized in 0.3% H2O2 for 15 min. After blocking, sections were incubated with p-Smad or t-Smad 1/5/8 antibodies (1:10) overnight at 4°C. Slides were incubated for an hour at RT with donkey anti-rabbit secondary antibody conjugated with horseradish peroxidase-labeled polymer (Jackson ImmunoResearch, West Grove, PA). Slides were incubated in TSA Plus Cyanine 3 (PerkinElmer, Waltham, MA) at 1:1500 for 30 min, Wheat Germ Agglutinin (WGA), Alexa Fluor® 488 (Invitrogen, Carlsbad, CA) at 1:200 for 10 min, followed by Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) at 1:20,000 for 10 min. The prepared slides were viewed under an Olympus DP 71 fluorescence microscope (Olympus, Center Valley, PA).
4
Immunostaining with Diverse Antibodies
5
Western Blot Analysis of Signaling Pathways
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Western blot was performed using TF1-BA and TF1-BAP cells treated with IM (2 M), AG490 (25 M) or E6201 (100 nM) during 24 hours (h), proteins were then extracted using RIPA buffer and 40 g of proteins were loaded. Membranes were incubated with monoclonal-antibodies against P-ERK (#4370), ERK (#4695), P-STAT3 (#9145), STAT3 (#9139), Caspase 3 (#9662). P-ABL (#2865), P-CRKL (#3181), P-Smad1/5/8 (#13820), GAPDH (#8884) (Cell Signalling Technology), BCR-ABL (ThermoFisher, #MA1-153) and Smad (SantaCruz Technology, #sc6031).21 (link)
6
Western Blot Analysis of Signaling Proteins
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The cells were lysed by ice-cold RIPA lysis buffer (Biyuntian, China) containing protease inhibitors, and the protein concentration was quantified using a BCA protein assay kit (Biyuntian). Each group of proteins was denatured by boiling, and then sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins. Subsequently, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) by electrophoresis. Then, the membranes were blocked in 5% skim milk for 1 h and incubated overnight at 4 °C in diluted primary antibody. Finally, images of the target strip were developed using a Western Chemiluminescent HRP Substrate Kit (Millipore, USA). Image Lab software (Bio-Rad, USA) was used for semi-quantitative analysis. Primary antibodies directed against AMPK (D5A2 for monoclonal antibody, Cell Signaling Technology), p-AMPK (40H9 for monoclonal antibody, Cell Signaling Technology), β-catenin (polyclonal antibodies, Cell Signaling Technology), p-β-catenin (polyclonal antibodies, Cell Signaling Technology), p-Smad-1/5/8(D5B10 for monoclonal antibody, Cell Signaling Technology), Smad-1/5/8 (N-18 for monoclonal antibody, Santa Cruz Biotechnology) and GADPH (6C5 for monoclonal antibody, Beyotime) were used.
7
Molecular Mechanisms of Hepatocellular Carcinoma
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Standard sugars were obtained from Sigma-Aldrich Chemical Company. Human hepatocellular carcinoma (HepG2) cells were obtained from the Tongji Medical College. Fetal bovine serum (FBS), DMEM medium, trypsin EDTA, penicillin, and streptomycin were obtained from Gibco (Grand Island, NY, USA). Anti-mouse β-actin antibody, horseradish peroxidase- (HRP-) conjugated anti-mouse, and anti-rabbit secondary antibodies were purchased from Sigma (St. Louis, MO, USA). Primary antibodies against STAT3, SMAD4, p-STAT3, p-SMAD1/5/8, Hepcidin, p-JAK2, SOCS3, BMP6, Id1, ferroportin, ferritin, IKKα, NF-κB, p-IκBα, Histone3.1, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA). The bicinchoninic acid (BCA) protein assay kit was purchased from Beyotime biotechnology (China). All the other reagents and chemicals were of analytical grade.
8
Embryonic Protein Expression Analysis
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Embryos were manually deyolked and harvested at the time points indicated. Western blot analyses for Ctsk were performed as described previously (Petrey et al., 2012 (link)). The western blots for pSmad2 and pSmad1,5,8 were blocked with 1% polyvinylpyrrolidone (PVP), followed by a 2-day incubation with the appropriate primary antibody (pSmad2 at 1/500, catalog no. 8828, Cell Signaling Technology; pSmad1,5,8 at 1/500, catalog no. 9511, Cell Signaling Technology). The western blots of Chst11 were performed using a zebrafish-specific antibody (1:1,000, catalog no. PA5–72647, Thermo Fisher Scientific, Rockford, IL). The blots were developed using the Bio-Rad Clarity substrate as directed and analyzed using the Bio-Rad MP Chemidoc system.
9
Pluripotent Stem Cell Protein Analysis
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The western blotting was performed using routine protocol as previously described 21 (link). Antibodies against Nanog, SOX2, OCT4, Smad 1, p-Smad 1/5/8 and GAPDH were purchased from Cell Signaling Technologies and used at 1:1000 dilution. ID1 antibody was purchased from Proteintech and used at 1:1000 dilution.
10
Molecular Profiling of Vascular Smooth Muscle
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