Irdye 800 conjugated affinity purified goat anti rabbit second antibody

After 14 days of odontoblastic differentiation, the total protein of cells was extracted by RIPA buffer (Beyotime Biotechnology, Haimen, China). The protein was electrophoresed and transferred to PVDF membrane. After PVDF membrane was blocked with 5% BSA, it was successively incubated with the primary antibody: rabbit anti-RUNX2 primary antibody (1:800, Abcam, Cambridge, UK), rabbit anti-DSPP (1:1000, Abcam, Cambridge, UK), rabbit anti-DMP-1 (1:1000, ABclonal, Wuhan, China), or mouse anti-β-actin (1:1000, Abcam, Cambridge, UK) and the second antibody: IRDye 700-conjugated affinity-purified goat anti-mouse (1:4000, Rockland Immunochemicals, Philadelphia, Pennsylvania, PA, USA) or IRDye 800-conjugated affinity-purified goat anti-rabbit second antibody (1:4000, Rockland Immunochemicals, Philadelphia, Pennsylvania, PA, USA). The relative protein expression level was analyzed with Odyssey laser scanning system (LI-COR Inc., Lincoln, NE, USA).

Western blot analysis was applied to determine the levels of protein expression in cells according to previous studies^{25 (link)–28 (link)}. Briefly, the proteins in the four groups were extracted by RIPA buffer (Beyotime, China) and transfected onto the PVDF membranes, then the PVDF membranes were incubated with 1:500 diluted mouse anti-p38 primary antibody (Abcam, UK), 1:500 diluted mouse anti-p-p38 primary antibody (Santa Cruz, USA), 1:500 diluted mouse anti-α-SM-actin primary antibody (Abcam, UK) and 1:1000 diluted rabbit anti-β-actin primary antibody (Sigma-Aldrich Co., USA) overnight at 4 °C. The following day, the membranes were probed with the 1:5000 IRDye diluted 700-conjugated affinity-purified goat anti-mouse second antibody (Rockland Immunochemicals, USA) or 1:5000 diluted IRDye 800-conjugated affinity-purified goat anti-rabbit second antibody (1:5000, Rockland Immunochemicals, USA) for 60 min at room temperature. Then protein bands were visualized using Odyssey laser scanning system (LI-COR Inc., USA) and the intensities were quantified by Odyssey 3.0 image analysis system software.