Investigating Osteoblast Signaling Pathways

MC3T3 cells were transfected with siRNA and/or stimulated with TNFα (10 ng/mL) for 1 hour. Whole-cell and nuclear lysates were prepared using RIPA buffer and isolation kit, respectively (Thermo Scientific, Rockford, IL, USA). Immunoblotting was carried out as described.^{(39 (link))} For some of the experiments, MC3T3 cells were preincubated with TNFα (10 ng/mL) for 45 minutes, followed by incubation with Bmp2/Wnt3a (100 ng/mL) for 4 hours. Some of the cells were then incubated with cycloheximide (10 μg/mL) for 3 hours. Nuclear and cytosolic lysates were prepared following the manufacturer’s recommendations (Thermo Scientific), and immunoblotting was carried out as described earlier using antibodies against β-catenin or Runx2 (Cell Signaling Technology, Beverly, MA, USA) according to the manufacturer’s recommendations.^{(35 (link))}