Anti dlx3

The expressions of Dlx3 and Osx in mandibular first molars of both wild type and Bmp2-cKO^od mice were analyzed by immunohistochemistry as described previously^{57 (link)}. MD10-F2 cells cultured on glass slides were fixed with cold methanol/acetone (1:1) for 10 minutes and permeabilized with 0.5% Triton-X for 15 minutes. To block the non-specific binding of antibodies, slides were incubated with 10% goat serum for 30 minutes, followed by primary antibodies, anti-Dlx3 (Abcam) and anti-Osx (Santa Cruz), overnight at 4^oC. After washing with PBS, slides were incubated with secondary IgG antibodies conjugated to Alexa-Fluor 568 (Invitrogen) were added and incubated for 1 hour. Hoechst (Pierce) was used to stain nucleus.

Cells were harvested and lysed for total protein in RIPA lysis buffer (Thermo Scientific, EU, Lithuania) with protease inhibitors. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were then incubated with anti-DLX3, anti-CyclinD1, anti-C-myc, anti-Tcf-7, anti-DKK1, anti-β-actin, anti-GAPDH (all from Abcam, Cambridge, MA, United States), and anti-active-β-catenin antibodies (Millipore, Burlington, MA, United States) overnight at 4°C. Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000) and chemiluminescence substrate were used to detect the immunoreactive bands with autoradiography film. Protein levels were quantitatively analyzed with Image J (National Institutes of Health, Bethesda, MD, United States) with β-actin as the loading control.