Psmad3

Manufactured by Merck Group

Sourced in United States, United Kingdom

PSMAD3 is a laboratory equipment product developed by Merck Group. It is a scientific instrument designed for specific functions within a laboratory setting. The core function of PSMAD3 is to perform tasks related to the analysis and processing of samples, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Smad3/pSmad3 (2 citations)

4 protocols using psmad3

Western Blot Analysis of SMAD2/3, Erk1/2

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WB analysis of SMAD2/3 and Erk1/2 was conducted as described [6 (link), 9 (link)], using primary antibodies against total SMAD2/3 (#3102, Cell Signaling), pSMAD2 (#3104, Cell Signaling), pSMAD3 (#07-1389, Merck Millipore), Erk1/2, pErk1/2 (#9102 and #9101; Cell Signaling Technology), β-actin (#A1978, Sigma-Aldrich) and α-tubulin (#2144; Cell Signaling Technology). The latter two were used as loading controls. Additional details are provided in Supplementary Materials.

Juste-Lanas Y., Díaz-Valdivia N., Llorente A., Ikemori R., Bernardo A., Arshakyan M., Borau C., Ramírez J., Ruffinelli J.C., Nadal E., Reguart N., García-Aznar J.M, & Alcaraz J. (2022). 3D collagen migration patterns reveal a SMAD3-dependent and TGF-β1-independent mechanism of recruitment for tumour-associated fibroblasts in lung adenocarcinoma. British Journal of Cancer, 128(6), 967-981.

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Transforming Growth Factor-β1 Signaling Pathway

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TGF-β1 was obtained from R&D Systems (Minneapolis, MN, United States). The following antibodies were purchased: BMP7 (Biosource, Camarillo, CA, United States), procollagen alpha 1 type 1 (1A1) (Santa Cruz Biotechnology, Santa Cruz, CA), p-Smad3, p-Smad1/5/8, p-P38 (EMD Millipore, Billerica, MA, United States), and glyceraldehyde 3-phosphate dehy-drogenase (GAPDH) (Research Diagnostics, Flanders, NJ, United States). Anti-α-SMA antibody, carbon tetrachloride (CCl₄), corn oil, and OptiPrep were purchased from Sigma-Aldrich (St. Louis, MO, United States). BMP7 was purchased from Selleckchem and Fisher Scientific (Waltham, MA, United States). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, United States) and Fisher Scientific (Waltham, MA, United States).

Zou G.L., Zuo S., Lu S., Hu R.H., Lu Y.Y., Yang J., Deng K.S., Wu Y.T., Mu M., Zhu J.J., Zeng J.Z., Zhang B.F., Wu X., Zhao X.K, & Li H.Y. (2019). Bone morphogenetic protein-7 represses hepatic stellate cell activation and liver fibrosis via regulation of TGF-β/Smad signaling pathway. World Journal of Gastroenterology, 25(30), 4222-4234.

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Western Blot Analysis of Aortic Proteins

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Aortic tissue homogenates were dissolved in sample buffer, run on a NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen, CA, United States), and transferred to nitrocellulose membranes using the iBlot transfer system (Invitrogen). Membranes were washed in PBS and blocked for 1 hr at room temperature with 5% instant non-fat dry milk dissolved in PBS containing 1% Tween-20 (Sigma, MO, United States) (PBS-T). Equal protein loading of samples was determined by a protein assay (Bio-Rad, CA, United States) and confirmed by probing with antibodies against β-Actin (Sigma). Membranes were probed overnight at 4° centigrade with primary antibodies against p Smad3 (1880-1; Millipore), Smad3 (#9513; Cell Signaling), p ERK1/2 (#4370; Cell Signaling), ERK1/2 (#4695; Cell Signaling), pPKCβ (#75837; Abcam, United Kingdom), PKCβ (#32026; Abcam), pPLCγ (#2821; Cell Signaling), and PLCγ (#2822; Cell Signaling), dissolved in PBS-T containing 5% milk. Blots were then washed in PBS-T and probed with HRP-conjugated anti-rabbit secondary antibody (GE Healthcare, United Kingdom) dissolved in PBS-T containing 5% milk at room temperature. Blots were then washed in PBS-T, developed using SuperSignalWest HRP substrate (Pierce Scientific, IL, United States), exposed to BioMax Scientific Imaging Film (Sigma), and quantified using ImageJ analysis software (NIH, MD, United States).

Doyle J.J., Doyle A.J., Wilson N.K., Habashi J.P., Bedja D., Whitworth R.E., Lindsay M.E., Schoenhoff F., Myers L., Huso N., Bachir S., Squires O., Rusholme B., Ehsan H., Huso D., Thomas C.J., Caulfield M.J., Van Eyk J.E., Judge D.P, & Dietz H.C. (2015). A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome. eLife, 4, e08648.

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Transcriptional Regulation by TGF-beta Signaling

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Cells were incubated overnight at 4°C with antibodies against: GATA‐1 (cat. #3535, Cell Signaling), pSmad2 (cat. #04‐953, Millipore), pSmad3 (cat. #52903, Abcam), tSmad3 (cat.#40854, Abcam), PU.1 (cat. #88082, Abcam), TIF1 (cat# 33475, Abcam) and TIF1γ (Cat# sc‐101179, santacrutz) all used at 1:1000 dilution and left overnight at 4°C. Detection was via a peroxidase‐conjugated anti‐rabbit antibody (cat. #111‐035‐003, Jackson Laboratories) or peroxidase‐conjugated anti‐mouse antibody (cat. # 115‐035‐003, Jackson Laboratories), incubated at a 1:20 000 dilution for 1 hour at RT. GAPDH (cat. #5174S, Cell Signaling) was used as a total cell extract control and Lamin B1 (cat. #13435S, Cell Signaling) as loading controls for nuclear extracts. Membranes were then incubated in ECL (cat. #80196, Pierce) for 5 minutes, washed and developed. Ligands were used at the following concentrations: GDF11 (100 ng/mL), activin B (10 ng/mL), GDF8 (100 ng/mL) and activin A (10 ng/mL) alone or in combination with luspatercept (1 μg/mL). HT‐1080 cell extracts treated with hTGFβ3 (cat# 12052S, Cell Signaling) serve as the positive control for pSmad2/3 in respective figures.

Martinez P.A., Li R., Ramanathan H.N., Bhasin M., Pearsall R.S., Kumar R, & Suragani R.N. (2020). Smad2/3‐pathway ligand trap luspatercept enhances erythroid differentiation in murine β‐thalassaemia by increasing GATA‐1 availability. Journal of Cellular and Molecular Medicine, 24(11), 6162-6177.

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