Protein Extraction and Western Blotting

Total protein was isolated from cultured primary chondrocytes, MSCs, or HEK293 cells after transfection using RIPA buffer supplemented with proteinase inhibitor cocktail (Sigma-Aldrich). Western blotting was performed as previously described^{47 (link)}, using anti-FLAG tag (M2, Sigma-Aldrich), anti-myc tag (TA150121, Origene), anti-HA (ab18181, Abcam), anti-Col1a1 (ab34710, Abcam), anti-Rankl (ab45039, Abcam), or anti-Gapdh (ab9485, Abcam) antibodies. All uncropped images were provided in the Supplementary data.

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Lui J.C., Raimann A., Hojo H., Dong L., Roschger P., Kikani B., Wintergerst U., Fratzl-Zelman N., Jee Y.H., Haeusler G, & Baron J. (2022). A neomorphic variant in SP7 alters sequence specificity and causes a high-turnover bone disorder. Nature Communications, 13, 700.

Publication 2022

proteinase inhibitor cocktail anti-FLAG tag (M2, ab18181 ab34710 ab45039 ab9485

Antibodies Buffer Chondrocytes Gapdh Hek293 cells Protein Proteinase inhibitor Rankl Ripa Transfection

Corresponding Organization : National Institutes of Health

Other organizations : Medical University of Vienna, The University of Tokyo, National Eye Institute, Ludwig Boltzmann Institute of Osteology, Hanusch Hospital

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Variable analysis

independent variables

Cell type (cultured primary chondrocytes, MSCs, or HEK293 cells)
Transfection

dependent variables

Total protein levels
Protein expression of FLAG tag, myc tag, HA, Col1a1, Rankl, and Gapdh

control variables

RIPA buffer supplemented with proteinase inhibitor cocktail (Sigma-Aldrich) for protein isolation
Western blotting protocol as previously described in reference 47

controls

Positive controls: not specified
Negative controls: not specified

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