Cells were lysed on ice in buffer containing 50mM HEPES, pH7.4, 80mM NaCl, 5mM MgCl2, 10mM EDTA, 5mM sodium pyrophosphate*10H2O, 1% TritonX-100, and Protease Inhibitor Cocktail (Sigma-Aldrich). 30 μg of total protein from each sample was resolved on Novex 4-20% Tris-Glycine Mini Protein Gels and transferred onto nitrocellulose membranes. The blots were probed with the appropriate antibodies: anti-ubiquitin (Cell Signaling Technology), anti-β-actin (Sigma-Aldrich), anti-20S/β1 subunit (Santa Cruz Biotechnology, Inc.), anti-20S/β2 subunit (Santa Cruz Biotechnology, Inc.), anti-20S/β5 subunit (Santa Cruz Biotechnology, Inc.), anti-E-cadherin (BD Biosciences), anti-fibronectin (BD Biosciences), anti-vimentin (Cell Signaling Technology), anti-phospho-Smad2 (Ser465/467) (Cell Signaling Technology), or anti-Smad2/3 (Cell Signaling Technology). Signals were detected using Odyssey infrared imaging system (LI-Cor Biosciences).
Anti phospho smad2 ser465 467
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-Smad2 (Ser465/467) is a laboratory reagent used to detect and analyze the phosphorylation of Smad2 protein at serine residues 465 and 467. This antibody can be used in various analytical techniques, such as western blotting, to study the activation and signaling pathways involving the Smad2 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti smad2 3, by Cell Signaling Technology (4 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti phospho smad3 ser423 425, by Cell Signaling Technology (2 mentions)
Anti smad2, by Cell Signaling Technology (2 mentions)
Anti ubiquitin, by Cell Signaling Technology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti fibronectin, by BD (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Ecl plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti c ebpβ, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Bioworld Technology (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti lamin b1, by Proteintech (1 mentions)
Anti smad4, by Cell Signaling Technology (1 mentions)
Anti c ebpβ, by Cell Signaling Technology (1 mentions)
Anti atad2, by Abcam (1 mentions)
9 protocols using anti phospho smad2 ser465 467
1
Protein Expression Analysis by Western Blot
2
Western Blotting Protocol for Otx2 and Smad Signaling
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Western blotting was performed as described previously (Yoshitomi et al., 2017 (link)). Briefly, cells were lysed in Laemmli buffer containing 5% 2-mercaptoethanol and were sonicated for 30 s and then heated at 98°C for 5 min. The resulting protein samples were subjected to SDS-PAGE using 4%–12% polyacrylamide gels (Wako, Osaka, Japan). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Invitrogen) and were blocked with 2% BSA in Tris-buffered saline. Membranes were then incubated with Anti-Otx2 antibody (cat. no. AF1979; R&D Systems, Minneapolis, MN), anti-Phospho-Smad2 (Ser465/467) (Clone no. 138D4, Cat. no. 3108, 1:1000, Cell Signaling Technology, Danvers, MA), anti-Phospho-Smad3 (Ser423/425) (Clone no. C25A9, Cat. no. 9520, 1:1000, Cell Signaling Technology), anti-Smad2/3 (Clone no. D7G7, Cat. no. 8685, 1:1000, Cell Signaling Technology), and anti-β-actin (Cat. no. A2228, 1:4000; Sigma-Aldrich) primary antibodies, followed by appropriate HRP conjugated secondary antibodies (Amasham GE, MA). Protein signals were then detected using ECL Plus Chemiluminescent Substrate (Thermo Fisher Scientific, Rockford, IL) with a Vilber-Lourmat FUSION FX7 imaging system (Vilber, Collégien, France).
3
Investigating TGF-β1 Signaling Pathway
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Antibodies used in this study are listed as follows: anti-ATAD2 (#ab176319, Abcam), anti-TGF-β1 (#sc-130348, Santa Cruz Biotechnology), anti-Smad2/3 (#8685, Cell Signaling Technology), anti-Smad4 (#46535, Cell Signaling Technology), anti-Phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (#8828, Cell Signaling Technology), anti-Phospho-Smad2(Ser465/467) (#18338, Cell Signaling Technology), anti-Phospho-Smad3(Ser465/467) (#ab52903, Abcam), anti-Snail (#3879, Cell Signaling Technology), anti-E-Cadherin (#3195, Cell Signaling Technology), anti-N-Cadherin (#13116, Cell Signaling Technology), anti-Vimentin (#5761, Cell Signaling Technology), anti-β-actin (#64132, Bioworld), anti-Lamin B1 (#66095–1-Ig, Proteintech), anti-C/EBPβ (#sc-7962, Santa Cruz Biotechnology), anti-C/EBPβ (#3087, Cell Signaling Technology).
The small-molecule TGF-β1 receptor inhibitors LY2157299 and SB525334 were purchased from Selleck Biochemicals.
The small-molecule TGF-β1 receptor inhibitors LY2157299 and SB525334 were purchased from Selleck Biochemicals.
4
Evaluation of HDAC Inhibitors and TGF-β Signaling
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Two kinds of class I and II HDACis, TSA and SAHA, were obtained from Union Biomed (Taipei, Taiwan); recombinant human TGF-β1 was obtained from R&D Systems (Minneapolis, MN, USA); the anti-ANKH and anti-α-tubulin antibodies were obtained from Novus Biologicals (Littleton, CO, USA); the class I HDAC antibody sampler kit, class II HDAC antibody sampler kit, anti-Smad2, anti-phospho-Smad2 (Ser465/467), anti-histone H3, anti-acetyl-histone H3-Lys9, and anti-acetylated-lysine (Ac-K-103) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA); the anti-ENPP1, anti-TNAP, anti-acetyl-histone H4, anti-TGF-β receptor I (TGFBR1), and anti-TGF-β receptor II (TGFBR2) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and the anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP), and anti-rabbit IgG HRP antibodies were obtained from GeneTex International (Hsinchu City, Taiwan).
5
Characterization of Activin A and VWC2 Proteins
6
Protein Expression and Quantification
7
Protein Extraction and Western Blot Analysis
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Whole-cell protein extract was prepared from LLC or Hep3B cells in RIPA buffer [10 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% (v/v) NP-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% protease inhibitor cocktail (Nacalai Tesque), and 1% phosphatase inhibitor cocktail (Nacalai Tesque)]. The extracted proteins were resolved on SDS–PAGE and transferred to an Immobilon-P transfer membrane (Millipore, Bedford, MA). The following antibodies were used: anti-phospho-Smad2 (Ser465/467) (1:1000), anti-Smad2 (D43B4) (1:1,000), anti-phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428), (1:1,000), anti-Smad1 (D59D7) (1:1,000), anti-Bcl-xL (54H6) (1:1000), anti-Bcl-2 (50E3) (1:1,000) (all from Cell Signaling Technology, Danvers, MA); anti-GAPDH (1:1,000) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-mLRG332 (1:500) (from IBL); and anti-human LRG (hLRG) polyclonal antibody (1:500) (from Proteintech Group, Chicago, IL). This was followed by treatment with 1:5,000 diluted donkey anti-rabbit horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-Sciences, Piscataway, NJ) and visualization using the Western Lightning ECL reagent (Perkin-Elmer, Boston, MA).
8
Quantification of SERPINE1 and TP53 in Cell Signaling
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All the antibodies used in the study were listed in Table 2 . Recombinant human GDF8 (788-G8), GDF8 Quantikine ELISA Kit (DGDF80), and Human SERPIN1 Quantikine ELISA Kit (DSE100) were purchased from R&D Systems (MN, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1706515) and goat anti-mouse (1706516) secondary antibodies and Clarity Max Western ECL Substrate (1705062) were obtained from Bio-Rad (CA, USA). SB-431542 (S1067) and U0126 (S1102) inhibitors were obtained from Selleck (Shanghai, China).
Antibody information
| Antibody name | Manufacturer (catalog number) | Applications (working dilution) |
|---|---|---|
| Anti-SERPINE1 | Proteintech (13801-1-AP) | WB (1:2,000) |
| Anti-TP53 | Proteintech (10442-1-AP) | WB (1:2,000) |
| Anti-α-Tubulin | Santa Cruz (sc-23948) | WB (1:5,000) |
| Anti-Lamin B1 | Santa Cruz (sc-374015) | WB (1:500) |
| Anti-phospho-SMAD2Ser465/467 | Cell Signaling (3108) | WB (1:1,000) |
| Anti-SMAD2 | Cell Signaling (3103) | WB (1:1,000) |
| Anti-phospho-SMAD3Ser423/425 | Cell Signaling (9520) | WB (1:1,000) |
| Anti-SMAD3 | Cell Signaling (9523) | WB (1:1,000) |
| Anti-phospho-p44/42 MAPK (Erk1/2)Thr202/Tyr204 | Cell Signaling (9106) | WB (1:2,000) |
| Anti-p44/42 MAPK (Erk1/2) | Cell Signaling (9102) | WB (1:2,000) |
MAPK, mitogen-activated protein kinase.
9
Phosphorylated SMAD2 Immunohistochemistry
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At 2 h after compression, samples were fixed over night at 4 C in 10% phosphate buffered formalin. Specimens were dehydrated and embedded in paraffin. 7 mm thick sections were cut and mounted on Superfrost™ Plus Microscope Slides (Thermo Scientific, USA). Then, the immunohistochemistry for c-terminally phosphorylated SMAD2P (rabbit mAb anti Phospho-Smad2 (Ser465/467) (1:100) (Cell Signalling Technology, Danvers, Massachusetts, USA)) was performed as previously described 21 .
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