Anti dspp

Cells were lysed in ice-cold radioimmunoprecipitation assay lysis and extraction buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocols. Whole-cell lysates were quantified using the bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). Protein samples were loaded and then transferred for immunoblotting. The membranes were incubated with the following primary antibodies overnight at 4 °C: anti-RUNX2, anti-collagen I, anti-RUNX2, anti-DSPP, anti-OPN, or anti-GAPDH antibody from Abcam Cambridge, UK, and Santa Cruz Biotechnology, Inc., Dallas, TX, USA. After washing, membranes were incubated with a secondary antibody from Santa Cruz Biotechnology, Inc. at room temperature.

The immunocytochemistry protocol has been reported previously [16 (link)]. Cells were seeded on poly-L-lysine-treated glass coverslips (8.000 cells/well). After reaching 30% confluence, the cells were differentiated for 21 days as described above. Monolayers were fixed with paraformaldehyde (4%), permeabilized, and incubated with one of two primary antibodies: first, polyclonal anti-DSPP (Abcam, Cambridge, MA, USA), which is specific to the DSPP N-terminus corresponding to the natural cleavage fragment dentin sialoprotein (DSP); second, anti-DMP-1 (Sigma-Aldrich). Both were prepared in blocking buffer at 1 : 50 dilution. Goat anti-rabbit IgG biotinylated antibody was added at room temperature, the samples were washed, and peroxidase-coupled streptavidin (Thermo Fisher Scientific) was added. Specific binding was visualized using H₂O₂ and 3,3′-diaminobenzidine tetrahydrochloride chromogen. Nuclei were counterstained with hematoxylin, and the cells were photographed using a Zeiss Axio Imager A2 microscope (Gottingen, Germany). Other culture sets were processed for immunofluorescence using Alexa Fluor 594 coupled streptavidin (Thermo Fisher Scientific). The nuclei were counterstained with Hoechst, observed in an Axio Imager A2 microscope (Zeiss, Germany) and analyzed with the AxioVision software.