Ab76956

The drilled tooth was prepared with RIPA cell lysis buffer, the supernatants were collected, and the total protein by the BCA kit (Thermo Scientific, Rockford, IL, United States of America) was measured. It was added 20 μg total protein per line to separate by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Then, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. PVDF membranes were blocked in TBS-T at room temperature for 2 h and washed three times. Next, they were incubated with primary antibodies at 4 °C overnight.
Primary antibodies included that BMP-2 (1:1,000, ab225898, Abcam), osteocalcin (1:2,000, ab76956, Abcam), osterix (1:1,000, ab209484, Abcam), and Runx2 (1:500, ab76956, Abcam), Wnt3a (1:1,000, ab219412, Abcam), p-GSK3β (1:1,000, #5558, CST), GSK3β (1:1,000, #12456, CST), p-β-catenin (1:1,000, #2009, CST), β-catenin (1:1,000, #8480, CST) and GAPDH (1:5,000; #5174, CST). Then, membranes were incubated with goat anti-mouse IgG (H+L) HRP (1:5,000, ab6789, Abcam) and goat anti-rabbit IgG (H+L) HRP (1:5,000, s0001, Affinity) secondary antibodies for 2 h at room temperature and washed three times. PVDF membranes were detected with ECL, and the protein bands were quantified by Scion Image 4.0 software (Scion Corporation, Frederick, MD, United States of America).

Total protein was extracted from cells and tissues with SDS lysis buffer (Beyotime), with the concentration measured utilizing a bicinchoninic acid kit (20201ES76, YEASEN Biotechnology Co., Ltd., Shanghai, China). After separation using 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the sample was submitted to electrotransfer onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) which were blocked using 5% blocking solution with skimmed milk powder and underwent overnight incubation at 4°C with primary rabbit antibodies against CGRP (ab189786, 1 : 1000, Abcam), RUNX2 (ab76956, 1 : 1000, Abcam), OCN (ab93876, 1 : 500, Abcam), ALP (ab229126, 1 : 1000, Abcam), CD45 (ab40763, 1 : 5000, Abcam), CD73 (ab133582, 1 : 5000, Abcam), and CD90 (ab92574, 1 : 1000, Abcam) as well as 1 h-incubation with horseradish peroxidase-labeled secondary antibody goat anti-rabbit IgG (ab150077, 1: 1000, Abcam) at ambient temperature. The immunocomplexes on the membrane were visualized utilizing enhanced chemiluminescence (ECL) reagent (ECL808-25, Biomiga, USA) at ambient temperature for 1 min, and band intensities were quantified with the help of Image Pro Plus 6.0 software (Media Cybernetics, Silver Springs, MD, USA).

Total proteins were obtained using an EpiQuik Total Histone Extraction Kit (Epigentek, Farmingdale, NY, U.S.A.; cat. no. OP-0006-100) according to instructions provided with the kit. Protein concentrations were determined using a Bradford Protein Assay Kit (Solarbio, Beijing, China; cat. no. PC0010). Aliquots of total protein from each sample were separated by 10% SDS-PAGE, and the protein bands were transferred onto PVDF membranes (Millipore, Burlington, MA, U.S.A.). The membranes were then blocked with 5% low fat dried milk for 2 h at room temperature and subsequently incubated with primary antibodies against RUNX2 (1:500, mouse, Abcam, Cambridge, U.K.; ab76956) and β-actin (1:3000, mouse, Abcam; ab20272) overnight at 4°C. After washing, the membranes were treated with Goat Anti-Mouse IgG (HRP, 1:5000, Abcam; ab205719) for 2 h at room temperature. The results were examined using enhanced chemiluminescent reagents.