Western blotting analysis was performed according to a standard method, as described previously [44 (link)], using anti–SMAD3, anti–Phospho-Smad3 (Ser423/425), anti–p21 Waf1/Cip1 and anti–c-MYC (Cell Signaling Tech, USA). Following the initial western blot assay, the membranes were stripped and re-probed with anti-GAPDH (Tianjin Sungene Biotech Co., China) as a protein loading control.
Anti phospho smad3 ser423 425
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-Phospho-Smad3 (Ser423/425) antibody is a highly specific and sensitive tool for detecting Smad3 phosphorylated at serine residues 423 and 425. This antibody is designed for use in various applications, including Western blotting, immunoprecipitation, and immunohistochemistry, to study the activation and signaling of the Smad3 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho smad2 ser465 467, by Cell Signaling Technology (2 mentions)
Anti p21 waf1 cip1, by Cell Signaling Technology (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Sungene Biotech (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Ecl plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti smad2 3, by Cell Signaling Technology (1 mentions)
Anti smad2 3 antibody, by Cell Signaling Technology (1 mentions)
Anti tgf β antibody, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti phospho smad1 5 ser463 465, by Cell Signaling Technology (1 mentions)
Anti p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Clarity max western ecl substrate 170 5062, by Bio-Rad (1 mentions)
Sb431542 s1067, by Selleck Chemicals (1 mentions)
Anti phospho gsk3β ser9, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Anti p70s6k, by Proteintech (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
6 protocols using anti phospho smad3 ser423 425
1
Protein Expression Analysis by Western Blotting
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
Western Blot Analysis of TGF-β Signaling
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Western blot analysis was performed as previously described [8 (link)]. The following primary antibodies were used: rabbit polyclonal anti-β-actin (Neomark, Fremont, CA), rabbit polyclonal anti-Smad2/3 antibody (Cell Signaling Technology, Inc., Boston, USA), rabbit polyclonal anti-phospho-Smad3 (Ser423/425; Cell Signaling Technology, Inc., Boston, USA), and anti-TGF-β antibody (Cell Signaling Technology, Inc., Boston, USA).
4
Antibody Validation for CHRDL2 and BMP Signaling
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Anti-CHRDL2 antibody (MAB2448) for western blot, anti-CHRDL2 antibody (AF2448) for immunohistochemistry, and HRP conjugate anti-mouse secondary antibodies (HAF007) were purchased from R&D. Anti-BMP2 (18933-1-AP), anti-BMP4 (12492-1-AP), anti-Activin A (60015-1-ig), and anti-BMP6 (60015-1-1g) antibodieswere obtained from Proteintech (China). Anti-Cyclin D1 (2978), anti-GAPDH (2118), anti-β-actin (12620), anti-Cleaved Caspase-9 (7237), anti-Cleaved Caspase-3 (9664), anti-Phospho-Smad1/5 (Ser463/465) (9516), anti-Smad1 (6944), anti-Smad4 (9515), anti-Smad2 (3122), anti-Phospho-Smad2 (Ser465/467) (11958), anti-Phospho-Smad3 (Ser423/425) (8828), anti-Smad3 (9513), anti-P21 (2947) and HRP Conjugate anti-rabbit secondary antibodies (7075) were obtained from Cell Signaling Technology Inc. (USA). Alexa Fluor 488 conjugated anti-rabbit secondary antibodies (P0176) and Alexa Fluor 555 conjugated anti-rabbit secondary antibodies (P0179) were obtained from Beyotime (China). Purified recombinant protein of Homo sapiens CHRDL2 (TP320245) and BMP2 (TP750012) were purchased from ORIGENE (USA).
5
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.
6
Protein Expression Analysis by Western Blot
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Cells seeded in 6-well plates were lysed by scraping into lysis buffer. The protein concentration of each lysate was examined using the BCA protein assay kit (TaKaRa, Japan). Western blotting was performed according to standard protocol with the following antibodies and dilutions: anti-KLF4 (Proteintech, #11880-1-AP, 1:1000), anti-CDH3 (Proteintech, #13773-1-AP, 1:500), anti-GSK-3β (Cell Signaling Technology, #12456, 1:1500), anti-Phospho-GSK-3β (Ser9) (Cell Signaling Technology, #9323, 1:1500), anti-AKT (Cell Signaling Technology, #9272, 1:1500), anti-Phospho-Akt (Ser473) (Cell Signaling Technology, #4051, 1:1500), anti-Erk1/2 (Thr202/Tyr204) (Cell Signaling Technology, #4695, 1:1500), anti-Phospho-Erk1/2 (Thr202/Tyr204) (Cell Signaling Technology, #4370, 1:1500), anti-p70S6K (Proteintech, #14485-1-AP, 1:500), anti-Phospho-p70 S6 Kinase (Cell Signaling Technology, #9204, 1:1500), anti-Smad3 (Cell Signaling Technology, #9513, 1:1500), anti-Phospho-Smad3 (Ser423/425) (Cell Signaling Technology, #9520, 1:1500), anti-β-catenin (Cell Signaling Technology, #8480, 1:1500) and anti-GAPDH (Proteintech, #60004-1-Ig, 1:10000).
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