G box f3 device

360,000 cells were differentiated in 6 cm dishes for 3 weeks in 1% FCS/OM containing the indicated substances or solvent control. Medium was changed every 2–3 days. Cells were lysed (20 mM Tris pH 7.5, 350 mM NaCl, 1% Triton X-100) for 20 min on ice. After centrifugation, protein concentrations in the supernatants were quantified with the DC protein assay (Bio-Rad). 5x Laemmli buffer (250 mM Tris pH 6.8, 500 mM DTT, 10% SDS, 0.5% Bromophenol blue, 35% Glycerol) was added, and samples were heated to 99 °C for 5 min. 50 µg total protein per lane were separated by SDS-polyacrylamid-gel-electrophoresis and electrotransferred to a PVDF membrane (GE Healthcare) following standard protocols. Blocking was performed with 10% BSA/TBS-T for 2 hours at room temperature. All antibodies were diluted in blocking solution: Osterix (abcam), Cbfa/Runx (MBL), Fibronectin (Santa Cruz), Collagen I (abcam) and Collagen IIa1 (Santa Cruz) 1:500; Osteopontin (abcam) 1:1000. GAPDH (hytest) served as a loading control and was applied at 1:100,000. After incubation with appropriate secondary antibodies (Dianova), SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) was used for development in a G:BOX F3 device (Syngene).

360,000 cells were incubated in 6 cm dishes for 3 weeks as indicated. Cells were lysed (20 mM Tris pH 7.5, 350 mM NaCl, 1% Triton X-100, 1 × Roche complete protease inhibitor cocktail, 1 mM PMSF, 1 mM sodium orthovanadate, 10 nM β-glycerophosphate, 5 mM NaF) for 20 min on ice. After centrifugation, protein concentrations in the supernatants were quantified with the DC protein assay (Bio-Rad). 5 × Laemmli buffer (250 mM Tris pH 6.8, 500 mM DTT, 10% SDS, 0.5% Bromophenol blue, 35% Glycerol) was added, and samples were heated to 99 °C for 5 min. 50 µg total protein per lane were separated by SDS-polyacrylamid-gel-electrophoresis and electrotransferred to a PVDF membrane (GE Healthcare) following standard protocols. Blocking was performed with 10% BSA /0.1% Triton X-100/TBS for 2 h at room temperature. All antibodies were diluted in blocking solution: Collagen I (abcam) and Cbfa/Runx (MBL) 1:500; Osteopontin (abcam) 1:1000. GAPDH (hytest) served as a loading control and was applied at 1:100,000. After incubation with appropriate secondary antibodies (Dianova), SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher) was used for development in a G:BOX F3 device (Syngene).