Immunoblotting was performed as described [14 (link)] with the following commercial primary antibodies: SMAD 1/5/9 (ab66737, Abcam, Cambridge, UK), total OXPHOS Antibody Cocktail (ab 110413, Abcam, Cambridge, UK), UCP1 (MAB6158, R&D System), β-tubulin (#2128), pSMAD 1/5/9 (#13820), Akt (#9272) and phospho-Akt (ser 473) (#9271) (all from Cell Signalling Technology, Danvers, MA, USA) and phospho-Akt (thr308) (#9275S BioLabs). Quantifications were performed by normalisation against loading controls.
P smad1 5 9
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States, China
The P-Smad1/5/9 is a laboratory equipment product developed by Cell Signaling Technology. It is designed to detect and quantify the phosphorylation of Smad1, Smad5, and Smad9 proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
P smad2, by Cell Signaling Technology (6 mentions)
Smad1 5 9, by Cell Signaling Technology (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Krt18, by Abcam (2 mentions)
Ap 2γ, by Abcam (2 mentions)
Brachyury t, by Novus Biologicals (2 mentions)
N cadherin, by Thermo Fisher Scientific (2 mentions)
E cadherin, by BD (2 mentions)
P smad1 5, by Cell Signaling Technology (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Ldn193189, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
34 protocols using p smad1 5 9
1
Immunoblotting Antibody Quantification Protocol
2
Western Blot Analysis of Osteogenic Markers
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Protein was extracted from MSCs and quantified as described above. Equal amounts of protein were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The polyvinylidene difluoride (PVDF) membranes were incubated with primary antibodies against GAPDH, OCN, Runx2, AKT, p‐AKT, Smad1, p‐Smad1/5/9, total catenin, p‐catenin, ERK1/2, and p‐ERK1/2 (all diluted 1:1000, Cell Signaling Technology) for 24 h and were then washed and incubated with a horseradish peroxidase (HRP)‐conjugated secondary antibody (1:3000, Cell Signaling Technology) for 1 h. Specific antibody‐antigen complexes were detected using Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Millipore).
3
Western Blot Analysis of Signaling Pathways
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The cell was collected and lysed after treatment, and membranes were incubated with primary antibodies as follows: anti-p-STAT3, p-Src, 3 hosphor-JAK2, p-RB, p-Smad1/5/9, STAT3, Src, JAK2, RB, Smad1/5/9, Survivin, Myc, IL-1β, IL-6, and β-tubulin were purchased from Cell Signaling Technology (Beverly, MA, USA). Membranes were probed with horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibody from Cell Signaling Technology. Protein bands were analyzed by a ChemiScope 6000Exp (CliNX, Shanghai, China). Quantification of band intensity was carried out using Image J software (NIH, Bethesda, Rockville, MD, USA).
4
Western Blot Analysis of Phosphorylated SMADs
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Protein concentration was quantitated using the BCA protein assay (Pierce) and 7.5 μg of protein was subjected to Western blot analysis. Membranes were blocked in EveryBlot blocking buffer (Bio-Rad; catalog 12010020) and then incubated with primary Abs against p-SMAD1/5/9 (1:750; Cell Signaling Technology; catalog 13820S), SMAD1 (1:1000; Cell Signaling Technology; catalog 6944S), or Actin (1:1000; Cell Signaling Technology; catalog 4970S), followed by incubation in HRP-conjugated secondary Abs (Cell Signaling Technology; catalog 7074S). Signals were detected with ECL Plus (Amersham Biosciences).
5
Investigating LKB1, p53, and RASG12D in Cancer
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LKB1 (#3050 for human, #3047 for mouse), p53 (Rodent specific, #32532), RASG12D (#14429), E-Cadherin (#3195), Vimentin (#3932), p-Smad1/5/9 (#13820), ID-3 (#9837) and AKR1C2 (#13035) antibodies were purchased from Cell Signaling Technology. BMP6 (ab155963), Smad 1/9 (ab108965), ID-1 (ab192303), Transferrin receptor (ab84036), SCD1 (ab236868), Ferritin (ab75973), EpCAM (ab71916) and TTF1 (ab76013) antibodies were from Abcam. KRAS (sc-30), p53 (sc-126 for human), a-Tubulin (sc-8035) antibodies were from Santa Cruz Biotechnology. Hepcidin Antimicrobial Peptide antibody (NBP1-59337) was from Novus Biologicals. Actin (A2066) was from Sigma-Aldrich. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch) were used for western blotting.
Neutralizing BMP6 antibodies (MAB507 for human, MAB6325 for mouse) were purchased from R&D system. LDN214117 (S7627) for in vitro and in vivo assay was from Selleckchem, and Cell Counting Kit-8 (CK04-11) was from Dojindo.
Neutralizing BMP6 antibodies (MAB507 for human, MAB6325 for mouse) were purchased from R&D system. LDN214117 (S7627) for in vitro and in vivo assay was from Selleckchem, and Cell Counting Kit-8 (CK04-11) was from Dojindo.
6
Western Blot Analysis of P-SMAD1/5/9
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For detection of the P‐SMAD1/5/9 protein level, cells were washed twice with PBS and lysed in 1xRIPA buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1% Nonidet P‐40, 1% Sodium Deoxycholic, 0.1% SDS), containing phosphatase and protease inhibitors (PhosSTOP cocktail and Complete tablets, Roche), NaF 50 mM and Na3VO4 1 mM. Protein concentration was determined by the PierceTM BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer's protocol and 15 µg of total lysates run onto precasted 4%–15% Midi Criterion TGX‐gels 18W (BioRad). Proteins were transferred onto Nitrocellulose membrane (BioRad) and probed with the indicated primary antibody at 4℃ overnight. After incubation with HRP‐conjugated secondary antibodies, protein bands were revealed by chemiluminescence with the Amersham ECL Detection Reagents (GE Healthcare) and detected with the Uvitec instrument (Cambridge, UK). Densitometric analysis of the western blot signals was performed by using the Uvitec software. Primary antibodies were diluted as follows: P‐SMAD1/5/9 1:2000 (#13820, SMAD1 1:2000 #9743, Cell Signaling); anti‐FLAG M2 monoclonal antibody 1:3000 (#F1804, Sigma‐Aldrich, Merk); anti‐GAPDH 1:20000 (#MAB374, Millipore).
7
Immunocytochemistry of Stem Cell and Differentiation Markers
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Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 3% bovine serum albumin for 30 min at room temperature. After blocking, cells were incubated overnight at 4 °C with primary antibodies. Primary antibodies are NANOG, TRA-1-81 (1:250, Stem cells); KRT8, KRT18, KRT19, AP-2γ, FOXG1 (1:200, Abcam); ALDH1A3, AP-2α, AFP (1: 200, Santa Cruz); Brachyury (T) (1:50, Novus); P63 (1:100, Genetex); p-SMAD1/5/9 (1:200, Cell Signaling); CDH1, Desmoplakin (1:500, BD Biosciences). Alexa 594-conjugated secondary antibody (1:400, red, Molecular Probes, Eugene, OR) or an Alexa 488-conjugated secondary antibody (1:400, green, Molecular Probes) was used to visualize the staining. Following three washes with PBS, slides were mounted with the VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA). Prior to mounting, slides were incubated with 2 μM 4′,6-diamidino-2-phenylindole (DAPI) fluorescence (Molecular Probes) for 10 min at 37 °C to stain the nuclei. The fluorescence images were taken using the EVOS FL Auto Cell Imaging System fluorescence microscope (ThermoFisher Scientific, NY, USA).
8
Immunostaining analysis of EMT markers
9
Phosphorylation Analysis of Lung Samples
10
Immunohistochemistry of Embryonic pSMAD1/5/9
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Embryos were fixed in 4% PFA for 2 hr on ice, washed in PBS and transferred to 30% sucrose overnight at 4°C. Embryos were frozen in OCT mounting medium, fixed to a chuck and cryosectioned immediately. Sections were dried for 2 hr and washed in PBS containing 0.1% triton for 10 min at room temperature. Sections were blocked in 0.1% Triton, 1 – 2% hings/serum for 1 – 1.5 hr at room temperature. Primary antibody solution was added (1:500 rabbit anti-PSMAD1/5/9 Cell Signalling Technology – D5B10) in PBS/0.1% triton/1 – 2% hings, before coverslips were added and slides placed in a humidified chamber at 4°C for 72 hr. Slides were then washed for 3 × 5 min in PBS at room temperature. Secondary antibody (1:500 anti-rabbit Alexa Fluor 594 Cell Signalling Technology – 8889S) was added in PBS 0.1% triton/1 – 2% hings for 1 hr at room temperature. Slides were washed in PBS for 3 × 5 min and mounted in Vectashield/DAPI medium. Slides were left at 4°C and imaged the following day.
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