Smad1 2 3

DPSCs were lysed and assayed with a BCA protein assay kit (Beyotime). Samples containing 15–30 μg of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins were then transferred to polyvinylidene fluoride membranes (Millipore). The membranes containing the transferred proteins were blocked with 5% bovine serum albumin and reacted with the primary antibody overnight at 4℃. The membranes were then labeled with corresponding secondary antibody of horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG for 1 h at room temperature before visualization with SuperSignal enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, US). Primary and secondary antibodies against the following proteins were used in this study: METTL3 (96391, 1:1000); METTL14 (51104, 1:1000); WTAP (56501, 1:1000); p-Smad1/5 (9516, 1:1000) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). NOG (sc-293439, 1:1000); RUNX Family Transcription Factor 2 (RUNX2, sc-390351, 1:1000); Dentin Sialophosphoprotein (DSPP, sc-73632, 1:1000); Smad1/2/3 (sc-7960, 1:1000); p-Smad3 (sc-517575, 1:1000) were obtained from Santa Cruz Biotechnology. GAPDH (60004-1-Ig, 1:3000); goat anti-mouse IgG (SA00001-1, 1:3000) and goat anti-rabbit (SA00001-2, 1:3000) were purchased from ProteinTech Group (ProteinTech, Wuhan, China).

At the end of the incubation periods, cultures were harvested, and equal amounts (5–15 μg) of whole cell lysates were subjected to electrophoresis in tris–glycine 4–15% gradient gels [28 (link)]. Membranes were incubated with the following primary antibodies: Type I collagen (1:400, Southern Biotechnology, Birmingham, AL), phospho-Smad2, phospho-FAK^S732, phospho-histone H1, phospho-CDK5 (1:1000, Cell Signaling Technology, Danvers, MA), total CDK5, Smad1/2/3, p35 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), and GAPDH (1:3000, Invitrogen), followed by appropriate secondary antibodies. Antigen–antibody complexes were visualized by chemiluminescence (Pierce Biotechnology, Rockford, IL). ImageJ software (http://rsb.info.nih.gov/ij/) was used to measure band intensities.