Alexa 568-conjugated Phalloidin (Invitrogen, Carlsbad, CA), SMA (Sigma-Aldrich) and Podoplanin (eBioscience, San Diego, CA) were used for immunofluorescence. Anti-TβRII (clone L21) and PAI-1 (Santa Cruz Biotechnologies, Santa Cruz, CA), Ki67 (Vector Laboratories, Burlingame, CA). and phospho-Smad2 (Cell Signaling, Danvers, MA) were used for immunohistochemistry. Antibodies used to detect protein by Western blotting were E-cadherin (BD Transduction, Franklin Lakes, NJ), vimentin (Sigma-Aldrich, St. Louis, MO); total Smad 2, phospho-Smad2, were purchased from Cell Signaling Technologies (Danvers, MA); Alpha-tubulin from Abcam (Cambridge, MA).
Phospho smad2
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Phospho-Smad2 is a laboratory product developed by Cell Signaling Technology. It is an antibody that specifically recognizes the phosphorylated form of the Smad2 protein, which is a key mediator in the transforming growth factor-beta (TGF-β) signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Phospho smad3, by Cell Signaling Technology (32 mentions)
Smad2, by Cell Signaling Technology (30 mentions)
Smad2 3, by Cell Signaling Technology (21 mentions)
Smad3, by Cell Signaling Technology (14 mentions)
Gapdh, by Cell Signaling Technology (13 mentions)
β actin, by Merck Group (13 mentions)
Phospho akt, by Cell Signaling Technology (11 mentions)
Phospho p38, by Cell Signaling Technology (11 mentions)
Gapdh, by Santa Cruz Biotechnology (9 mentions)
Vimentin, by Cell Signaling Technology (8 mentions)
β actin, by Cell Signaling Technology (8 mentions)
Smad4, by Cell Signaling Technology (7 mentions)
Snail, by Cell Signaling Technology (7 mentions)
N cadherin, by Cell Signaling Technology (7 mentions)
E cadherin, by Cell Signaling Technology (7 mentions)
α tubulin, by Merck Group (7 mentions)
Phospho erk1 2, by Cell Signaling Technology (6 mentions)
Total smad2, by Cell Signaling Technology (5 mentions)
β actin, by Santa Cruz Biotechnology (5 mentions)
Phospho smad1 5, by Cell Signaling Technology (5 mentions)
109 protocols using phospho smad2
1
Immunofluorescence and Immunohistochemistry Protocols
2
Immunofluorescence and Immunohistochemistry Protocols
3
Immunohistochemistry of Fibrotic Markers
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Hippuric acid was purchased from Sigma Aldrich (Cat. No. 112003). Antibodies were obtained for CD68 (Abcam; ab31630), phospho-SMAD2 (Cell Signaling Technologies; Cat. No. 3108), SMAD2 (Cell Signaling Technologies; Cat. No. 3103), transforming growth factor-β (TGF-β) (Cell Signaling Technologies; Cat. No. 3711), and collagen 1A (COL1A) (Cell Signaling Technologies; Cat. No. 91144). Harris Hematoxylin was purchased from Newcomer Supply (Cat. No. 1201). Xylene was purchased from Fisher Scientific (Cat. No. 05082-4). Eosin-Phloxine Working Solution was obtained from Newcomer Supply (Cat. No. 1082). Glacial acetic acid was purchased from Newcomer Supply (Cat. No. 10010 A). Sirius Red F3B (Cat. No. 36-554-8), a saturated aqueous solution of picric acid (Cat. No. P6744), and solid picric acid (Cat. No. 239801) were purchased from Sigma. Histoclear was obtained from National Diagnostics (Cat. No. HS-200). The DAB Staining kit (Ref: K4065), Protein Block reagent (Ref: X0909), and Antibody Diluent (Ref: 80809) were purchased from Dako. Cytoseal XYL Mounting Media was purchased from Thermo Scientific (Ref: 8312-4).
4
Anthocyanidins Modulate Epithelial-Mesenchymal Transition
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Anthocyanidin compounds Cy (purity ≥ 96%), Dp, Pg, Mv (purity ≥ 97%) and Pt (purity ≥ 95%) were from Extrasynthese (Lyon, France). Recombinant human TGF-β1 was from R&D Systems (Minneapolis, MN). Electrophoresis reagents were from Bio-Rad (Mississauga, ON). The anti-Twist1 monoclonal antibody was from Santa Cruz Biotechnologies (Dallas, TX). The anti-Periostin antibody was from Abcam (Cambridge, MA). Antibodies against Snail, phospho-Smad2, Smad2, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), vimentin, β-Catenin, phospho-Fak, Fak, phospho-Akt and Akt were from Cell Signaling Technology (Beverly, MA). The anti-fibronectin antibody was from Sigma Aldrich (Oakville, ON). Anti-mouse and anti-rabbit horseradish peroxidase (HRP)-linked secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA) and enhanced chemiluminescence (ECL) reagents were from Denville Scientific Inc. (Metuchen, NJ). Micro bicinchoninic acid protein assay reagents were from ThermoFisher Scientific (Rockford, IL).
5
Antibody Sourcing for Cell Signaling
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Antibodies (Abs) against phospho-Jnk1/2, total-Jnk1/2, phospho-PKB (pS473), total-PKB, phospho-p38, total-p38, phospho-Smad2, total-Smad2, phospho-Smad3, total-Smad3, PCNA, and α-tubulin were purchased from Cell Signaling (Beverly, MA, USA). An antibody against Gadd45β was obtained from Aviva Systems Biology (San Diego, CA, USA). Antibodies against β-actin, HA, Myc, and GST were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against V5 and Flag were purchased from Invitrogen (Carlsbad, CA, USA). Cy3-conjugated donkey anti-mouse IgG and Alexa 488-conjugated goat anti-rabbit IgG antibodies were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and Invitrogen (Waltham, MA, USA), respectively. An anti-Strep MAB-classic antibody and Strep-Tactin Sepharose were purchased from IBA (Gottingen, Germany). Sepharose 6B and Glutathione 4B were obtained from GE Healthcare (Little Chalfont, UK). Human recombinant TGF-β1 and an anti-BrdU monoclonal antibody were purchased from Sigma (St. Louis, MO, USA). Dextran sulfate sodium (DSS; M.W. = 36–50 kDa) was obtained from MP Biomedicals (Santa Ana, CA, USA).
6
Antibody Immunoblotting and IHC Protocol
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The following antibodies were used in this study: DCP1A (Santa Cruz, 56-Y), SYK (Santa Cruz, N19), G3BP1 (BD Biosciences, 611126), LC3-A/B (Cell Signaling Technology, D3U4C), p62 (Abcam, ab56416), E-cadherin for immunoblot (Santa Cruz, H10), DDX6 (Sigma Aldrich, P0067), GAPDH (Ambion, AM4300), SMAD2/3 (Cell Signaling Technology, 3102), phospho-SMAD2 (Cell Signaling Technology 3101), E-cadherin for IHC (BD biosciences, 610182), Ki67 (BD biosciences, 550609), AlexaFluor 594-conjugated goat anti-rabbit IgG (Invitrogen), AlexaFluor 594-conjugated goat anti-mouse IgG (Invitrogen), and AlexaFluor 488-conjugated goat anti-mouse IgG (Invitrogen), biotin-conjugated goat anti-mouse IgG (Jackson). For immunohistochemistry biotinylated secondary antibodies were detected using the ABC elite kit in combination with 3-3′ -diaminobenzidine (Vector).
7
Protein Extraction and Western Blotting
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Protein extraction and Western blotting experiments were performed as described previously [12 (link)]. Primary antibodies used were as follow: anti-TGF-β1, CDK4, c-Myc, CyclinD3, p27, phospho-Akt, Akt, phospho-Erk1/2, Erk1/2, phospho-JNK, JNK, phospho-p38, p38, phospho-Smad 2, Smad 2/3, Smad 4, and Histone H3 (Cell Signaling, Danvers, MA); anti-CD63, CD81, and CD9 (Santa Cruz, Santa Cruz, CA); and anti-β-actin (Sigma-Aldrich). HRP-conjugated horse anti-mouse and goat anti-rabbit IgG (Cell Signaling) were used as secondary antibodies.
8
Fibroblast Cell Culture and TGF-β Signaling
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Human fetal lung fibroblast (MRC5) cell lines were propagated in EMEM media (Gibco, Rockville, IL, USA) with 10% FBS (Hyclone, Logan, UT, USA), and 1% penicillin/streptomycin antibiotic mix (Lonza, Allendale, NJ, USA). The cells were kept in a 37 °C humidified incubator with 5% carbon dioxide. Immobilized protein A/G beads, FN, α-SMA, TβRI, HA tag, USP11, V5 tag, TβRII, and control IgG antibodies, USP11 siRNA (pools of three to five siRNA), and control siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-SMAD2, phospho-SMAD3, total SMAD2, SMAD3, and ubiquitin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Bleomycin, leupeptin, CHX, MTX, and antibodies against Flag-tag and β-actin were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant TGF-β1 was purchased from Invitrogen (Carlsbad, CA, USA). Proteasome inhibitor MG-132 was from Calbiochem (KGaA, Darmstadt, Germany). DAPI was purchased from ThermoFisher Scientific (Waltham, MA, USA). All materials in highest grades used in the experiments are commercially available.
9
Western Blot Analysis of Signaling Pathways
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For each sample, 20 μg of protein was separated by SDS/PAGE and transferred to PVDF membranes.30 The membranes were blocked for 1 hour at room temperature with 5% powdered skim milk in Tris‐buffered saline pH 7.4 with 0.05% Tween (TBST) and incubated with antibodies for TGFBR2 (#SC400; 1:1000; Santa Cruz Biotechnology, Dallas, TX), phospho‐SMAD2 (S465‐467, #3101S; 1:1000; Cell Signaling, Danvers, MA), SMAD2 (#3122S; 1:1000; Cell Signaling), phospho‐ERK1/2 (T202‐Y204, #9101S; 1:1000; Cell Signaling), ERK1/2 (#9102S; 1:1000; Cell Signaling), phospho‐p38 (T180‐Y182, #4511S; 1:2000; Cell Signaling), p38 (#9212S; 1:2000; Cell Signaling), or β‐actin (#A5316; 1:5000; Sigma, St. Louis, MO). Antibodies were diluted in TBST with 5% milk and incubated overnight at 4°C. Blots were then washed 3 times (5 minutes each) in TBST and incubated with HRP‐conjugated secondary antibody (goat antirabbit or antimouse; Biorad, Hercules, CA; #170‐6515 or #170‐6516; 1:3000‐1:5000 in TBST with 5% milk) for 1 hour at room temperature. Bound antibodies were detected using enhanced chemiluminescence (ECL) (Thermo, Rockford, IL). Band density was analyzed by densitometry, using the Image J software Version 1.48 (NIH, Bethesda, MD).
10
Comprehensive IHC and IF Profiling of Cell Markers
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Basic IHC protocols have been described previously [42 (link)]. Primary antibodies for E-Cadherin (1:100 dilution; Abcam), vimentin (1:50 dilution; Abcam), N-cadherin (1:50 dilution; Abcam), cytokeratin 8 (CK8) (1:50 dilution, Abcam), cytokeratin 5 (CK5) (1:50 dilution, Abcam), CD44 (1:300 dilution; Abcam), TGFβ1 (1:100 dilution; Santa cruz) and phospho-Smad2 (1:100 dilution; Cell Signaling) were used for this study. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide, and the sections were incubated with diluted antibodies. Slides were then incubated with various primary antibodies followed by Envision-plus labeled polymer-conjugated horseradish peroxidase and DAB monitoring staining (Zhongshan gold bridge, Beijing). To perform immunofluorescence experiments after deparaffinization, hydration and antigen retrieval as IHC protocol, slides were incubated with various primary antibodies followed by FITC or TRITC labeled second antibody and DAPI staining (Abcam). Finally, slides were viewed and imaged without dehydration.
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