For immunofluorescence analysis, SMSCs were seeded onto 24-well plates at 1 × 104 cells per well and then treated with tFNAs (0, 250 nM). After 24 h of treatment, the cell samples were rinsed with PBS three times and fixed in cold 4% formaldehyde solution for 20 min. After 3 washes with PBS again, Triton X-100 (0.5%) was used to permeabilize the SMSCs for 30 min, and then, the samples were blocked with immune blocking solution (Beyotime, Shanghai, China) for 30 min. Fixed SMSCs that were rinsed with PBS were then incubated with anti-β-catenin (1:200, Abcam, Cambridge, England), anti-Lef-1 (1:200, Novus, NY, USA), anti-cyclin D1 (1:200, Novus, NY, USA), anti-phospho-Smad2 (1:2000, Cell Signaling Technology, MA, USA), and anti-phospho-Smad3 (1:200, Cell Signaling Technology, MA, USA) primary antibodies overnight at 4 °C. Fluorescence-conjugated secondary antibody was then combined with primary antibodies for 1 h at 37 °C. DAPI (1:200; Beyotime, Shanghai, China) was applied to stain the cell nucleus for 10 min, and phalloidin (1:60; Beyotime, Shanghai, China) was used to stain F-actin for 40 min. Finally, cell images were observed by fluorescence microscopy (Keyence, Osaka, Japan).
Anti phospho smad3
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-Smad3 is a primary antibody that specifically recognizes the phosphorylated form of Smad3, a key intracellular signaling molecule involved in the transforming growth factor-beta (TGF-β) signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti smad3, by Cell Signaling Technology (21 mentions)
Anti phospho smad2, by Cell Signaling Technology (19 mentions)
Anti smad2, by Cell Signaling Technology (7 mentions)
Anti β actin, by Merck Group (7 mentions)
Anti phospho p38, by Cell Signaling Technology (4 mentions)
Anti phospho erk, by Cell Signaling Technology (4 mentions)
Anti smad1, by Cell Signaling Technology (3 mentions)
Anti smad4, by Cell Signaling Technology (3 mentions)
Anti p38, by Cell Signaling Technology (3 mentions)
Anti phospho jnk, by Cell Signaling Technology (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Anti smad2 3, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Recombinant human tgf β1, by R&D Systems (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti α sma, by Abcam (2 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti fibronectin, by Proteintech (2 mentions)
37 protocols using anti phospho smad3
1
Immunofluorescence Analysis of SMSC Signaling
2
Molecular Mechanisms of TGF-β1-Mediated Apoptosis
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Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA), emodin was obtained from Shanghai future industry Limited by Share Ltd (Shanghai, China) and BLM was acquired from Nippon Kayaku (Tokyo, Japan). The primary antibodies described in the study include: anti-E-cadherin, anti-vimentin, anti-cleaved caspase-3, anti-phospho-Smad2, anti-phospho-Smad3, anti-Smad2, anti-Smad3, anti-phospho-Erk1/2 and anti-Erk1/2 (Cell Signaling Technology, CA, USA); anti-caspase-3, anti-Bax (Santa Cruz Biotechnology, CA, USA); anti-fibronectin (Proteintech, Chicago, USA); anti-caspase-8, anti-Bcl-2 (Absci, MD, USA); anti-TGF-β1, anti-FSP-1, anti-α-SMA (Abcam, USA); and anti-GAPDH (Beyotime Institute of Biotechnology, Haimen, China). Other reagents were obtained from Beyotime Institute of Biotechnology unless otherwise indicated.
3
Whole-Cell Lysate Preparation for Western Blotting
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For preparation of whole-cell lysates, the cells were washed with ice-cold phosphate buffered saline (PBS) and lyzed for 30 min on ice in radio-Immunoprecipitation assay (RIPA) buffer with 150 mM NaCl as described.[7 (link)] Cell lysates were cleared at 20,000g for 10 min. After the adjustment of protein concentration, the lysates were boiled in sodium dodecyl sulfate (SDS) sample loading buffer for 5 min and separated by SDS–polyacrylamide gel electrophoresis. The gels were blotted on a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA, USA) and stained with the indicated first antibody [anti-phospho-Smad3 or anti-total-Smad3 (1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA)]. Antibody binding was detected with horseradish peroxidase-coupled secondary antibody followed by chemoluminescence detection (ECL Plus; Amersham Pharmacia, Uppsala, Sweden).
4
Characterization of Macrophage Phenotypes
5
Molecular Mechanisms of Glioblastoma Response to Temozolomide
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Reagent and antibody sources were as follows: AG1478 (Calbiochem/Merck, Darmstadt, Germany), BMP4 (R&D Systems, Minneapolis, MN, USA), DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), temozolomide (TMZ) and anti-β-Actin-peroxidase conjugated antibody (Sigma-Aldrich, Munich, Germany), anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-AKT (Thr308), anti-BIM, anti-cleaved caspase 3, anti-cleaved caspase 7, anti-cleaved PARP (poly (ADP-ribose) polymerase-1), anti-EGF Receptor, anti-phospho-EGF receptor (Tyr1068), anti-FOXO3a, anti-phospho-FOXO3a (Thr32), anti-phospho-FOXO3a (Ser253), anti-phospho-FOXO3a (Ser318/321), anti-phospho-Rb (Ser807/811), anti-SMAD1, anti-SMAD3, anti-SMAD4, anti-SMAD5, anti-phospho-SMAD1/5 (Ser463/465), anti-phospho-SMAD3 (Ser423/425), anti-p27Kip1, anti-SOX2 (Cell Signaling Technology, Beverly MA, USA), anti-OLIG2, anti-β-Tubulin beta III isoform (Millipore, Temecula, CA, USA), anti-CYCLIN B1, p21CIP1 (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-GFAP (BD Pharmingen San Jose, CA), anti-NESTIN (R&D Systems, Minneapolis, MN, USA), and anti-CYCLIN D1 (ThermoFisher Scientific, Waltham, MA USA).
6
Antibody Sourcing for Extracellular Matrix Research
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Anti-pro-collagen α1(1) N-propeptide (pN-Col1 α1) and anti-MMP1 antibodies were obtained as described [52 (link)]. We sourced antibodies from their respective suppliers: anti-SPARC (15274-1-AP; Proteintech, Chicago, IL, USA); anti-GAPDH (ab83108; AbClone, (Seoul, Republic of Korea); anti-SMAD2 (#5339), anti-phospho-SMAD2 (#3108), anti-SMAD3 (#9523), and anti-phospho-SMAD3 (#9520) (all from Cell Signaling Technology, Danvers, MA, USA); anti-collagen type I cleavage site (immunoGlobe Antikörpertechnik GmbH, Himmelstadt, Germany); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, goat anti-rabbit IgG, Alexa Fluor® 488 goat anti-rabbit IgG (H+L), and rhodamine Red X-conjugated goat anti-mouse IgG (Thermo Fisher Scientific Inc., Waltham, MA, USA) [53 (link)].
7
Protein Quantification and Validation Methodology
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Aliquots of cell lysate (50 µg/lane) were separated by electrophoresis on 10% or 12% SDS polyacrylamide gels. Anti-phospho-STAT6, anti-STAT6, anti-phospho-SMAD2 (#3104), anti-SMAD2, anti-phospho-SMAD3, anti-SMAD3, anti-phospho-SMAD1, 5, and 9, anti-SMAD1, 5, and 9, anti-phospho-ERK1/2 (44/42 MAPK) (#4370), anti-ERK1/2 (#9102), anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, and anti-SDHA (#5893) antibodies were purchased from Cell Signaling Technology (Beverley, MA, USA). Anti-NDUFA9 and COX-4 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-UQCRC2 (ab14745) was purchased from Abcam (Cambridge, UK). Anti-ATP5A1 was purchased from Invitrogen (Thermo Fisher). Secondary antibodies (goat anti-mouse and goat anti-rabbit) were obtained from Santa Cruz Biotechnology. Images were scanned on an ODYSSEY instrument and quantified using Image Studio Digits (LI-COR Biosciences, Lincoln, NE, USA). Full length uncropped blots are presented in Supplementary Figures 12 –17 .
8
Immunoblot Analysis of NF-κB Pathway Components
9
Transforming Growth Factor-β1 Regulation
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TGF-β1 was purchased from R&D Systems (Minneapolis, MN). Human has-miR-20a mimics and control miRNA mimics were from Thermo Scientific Dharmacon (Lafayette, CO). Antibodies were purchased as follows: anti-Smad2, anti-Smad3, anti-phospho-Smad2 and anti-phospho-Smad3 from Cell Signaling (Denver, MA); anti-p21CIP1, anti-Smad4, anti-c-Myc and anti-TβRII from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Ki67 from VECTOR (Burlingame, CA); and anti-β-actin from Sigma Biochemicals (St Louis, MO). The TRKI (LY2109761) was kindly provided by Dr. Jonathan Yingling (Eli Lilly Pharmaceuticals, Indianapolis, IN).
10
TGF-β1 Signaling Modulation Assay
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Recombinant human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). HCQ sulfate was purchased from Thermo Fisher Scientific (Waltham, MA, USA). CQ phosphate was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Compound C was purchased from Calbiochem (San Diego, CA, USA). Anti-phospho-AMPK, anti-AMPK, anti-LC3, anti-phospho-Smad2, and anti-phospho-Smad3 antibodies were from Cell Signaling Technology (Danvers, MA, USA), and anti-p27 and anti-Smad3 antibodies were from Santa Cruz Biotechnology. Anti-Cyclin D and anti-Collagen I were from Merck-Millipore (Temecula, CA, USA). anti-Collagen III was from Fitzgerald Industries International (Acton, MA, USA). Anti-tubulin was from Sigma-Aldrich (St. Louis, MO, USA).
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