Western Blot Analysis of Phospho-Smad3 Signaling

Cultured and treated cells were lysed in radioimmunoprecipitation assay buffer and subjected to Western blot analysis, as described in the previous report (Kang et al., 2018 (link)). The following primary and secondary antibodies were used for Western blotting: phospho-Smad3 (Ser423/425; C25A9) rabbit mAb (#9520; Cell Signaling Technology), phospho-Smad3 (Ser423/425) rabbit polyclonal Ab (#600–401-919; Rockland Inc.), Smad2/3 (D7G7) XP rabbit mAb (#8685; Cell Signaling Technology), SMAD3 (C67H9) rabbit mAb (#9523; Cell Signaling Technology), β-Actin (AC-15) mouse mAb (#A5441; Sigma), GAPDH (D16H11) XP rabbit mAb (#5174; Cell Signaling Technology), histone H3 (D1H2) XP rabbit mAb (#4499; Cell Signaling Technology), and FLAG (M2) mouse mAb (#F1804; Sigma). Secondary antibodies (LI-COR Biosciences) were IRDye 680RD anti-rabbit IgG (#926-68071) or IRDye 800CW anti-mouse IgG (#926-32210).

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Kang H., Jha S., Ivovic A., Fratzl-Zelman N., Deng Z., Mitra A., Cabral W.A., Hanson E.P., Lange E., Cowen E.W., Katz J., Roschger P., Klaushofer K., Dale R.K., Siegel R.M., Bhattacharyya T, & Marini J.C. (2020). Somatic SMAD3-activating mutations cause melorheostosis by up-regulating the TGF-β/SMAD pathway. The Journal of Experimental Medicine, 217(5), e20191499.

Publication 2020

Smad2/3 (D7G7) XP rabbit mAb SMAD3 (C67H9) rabbit mAb GAPDH (D16H11) XP rabbit mAb histone H3 (D1H2) XP rabbit mAb FLAG (M2) mouse mAb IRDye 680RD anti-rabbit IgG IRDye 800CW anti-mouse IgG

Actin Anti igg Antibodies Buffer Cells Gapdh Histone h3 Irdye 800cw Mouse Rabbit Radioimmunoprecipitation assay Smad2 Smad3 Western blot analysis

Corresponding Organization : National Institutes of Health

Other organizations : National Institute of Arthritis and Musculoskeletal and Skin Diseases, Allgemeine Unfallversicherungsanstalt, Hanusch Hospital, Ludwig Boltzmann Institute of Osteology, Office of Science, National Institutes of Health Clinical Center

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

Protein expression levels of phospho-Smad3 (Ser423/425), Smad2/3, SMAD3, β-Actin, GAPDH, and histone H3

control variables

Not explicitly mentioned

controls

Positive controls: None mentioned
Negative controls: None mentioned

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