Odyssey version 4

The fibroblasts were seeded with 150.000 cells/well and treated for 60 min with Activin A or 90 min with BMP4 after serum starvation overnight. Whole-cell lysates were prepared by lysing cells in NuPAGE^® LDS Sample Buffer with 10% NuPAGE^® reducing agent. Proteins were separated in NuPAGE 4–12% BT gels using the XCell SureLock™ system and were subsequently transferred by using the iBlot Dry Blotting system (Invitrogen). Nitrocellulose membranes were blocked in the Odyssey blocking buffer (Westburg). Immunoblotting was performed in Odyssey blocking buffer with 0.1% Triton X-100.
Primary antibodies against phospho-SMAD3 (cat#52903, Abcam), phospho-SMAD1/5/9 (AB3848-I, Sigma-Aldrich), and Actin (Cat#ab14128, Abcam) were used for overnight incubation. Secondary antibody incubation was carried out for 1 h with the IRDye 800 CW goat anti-rabbit IgG and IRDye 680 CW goat anti-mouse (LI-COR Biosciences). Fluorescence was visualized by the Odyssey system equipped with the Odyssey version 4 software (LI-COR Biosciences).

Transdifferentiated primary fibroblasts and primary human osteoblasts were lysed in NuPAGE® LDS Sample Buffer with NuPAGE® reducing agent by scraping. The whole cell lysates were subjected to electrophoresis after which proteins were transferred to a nitrocellulose membrane with the iBLOT transfer system (Invitrogen). After incubation in blocking buffer for 1 h (LI-COR Biosciences), the nitrocellulose membrane was incubated with the primary antibodies against RUNX2 (abcam; Cat#ab23981) and actin (abcam; Cat#ab14128) overnight at 4 °C. Visualization of the signal was performed after incubation with the secondary antibodies IRDye 800 CW goat anti-rabbit IgG and the IRDye 680 CW goat anti-mouse IgG antibodies by using the Odyssey infrared imaging system equipped with the Odyssey version 4 software (LI-COR Biosciences). The relative RUNX2 expression is quantified using Image Studio Lite (LI-COR Biosciences).

For the western blot analysis, PLF was cultured in 6 well plates, 3 × 10⁵ cells/well. After overnight culture under serum‐free conditions, cells were incubated without or with 50 ng/ml Activin‐A for 1 hr. Whole cell lysate preparation and western blot analysis was performed as described before (Micha et al., 2016). Shortly, cells were lysed using the NuPAGE LDS Sample Buffer with the NuPAGE reducing agent (Thermo Fisher Scientific, Waltham, MA). Proteins were separated on NuPAGE 4–12% BT gels and were subsequently transferred to nitrocellulose membranes using the iBlot Dry Blotting system (Thermo Fisher Scientific). Immunoblotting was performed overnight with primary antibodies against phosphoSmad3 (Abcam, Cat#ab52903) and actin (Abcam, Cat#ab14128). Secondary antibody incubation was carried out for 1 hr with the IRDye 800CW goat anti‐rabbit IgG and the IRDye 680CW goat anti‐mouse IgG antibodies (LI‐COR Biosciences, Lincoln, NE). Fluorescence was visualized and quantified by the Odyssey infrared imaging system equipped with the Odyssey version 4 software (LI‐COR Biosciences).