Sc 73633

Total protein was extracted from the HDPCs or pulp tissue samples using RIPA medium containing phenylmethanesulfonyl fluoride (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were determined using a BCA Protein Assay kit (P0012; Beyotime Institute of Biotechnology). Equivalent amounts of diluted protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. After blocking for 1 h at room temperature in 5% skim milk solution, the membranes were subsequently incubated with anti-TRPC6 antibodies (1:500; ab62461; Abcam, Cambridge, UK), anti-dentin sialophosphoprotein (anti-DSPP) antibodies (1:500; sc73632; Santa Cruz Biotechnologies, Inc., Dallas, TX, USA), anti-dentin matrix protein-1 (anti-DMP-1) antibodies (1:500; sc73633; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Secondary antibody anti-mouse IgG/anti-rabbit IgG, HRP-linked antibody (7076S/7074S; Cell Signaling Technology, Inc., Danvers, MA, USA) was then added at a dilution of 1:5,000 for 1 h at room temperature after the membranes were washed with Tris-buffered saline with Tween-20. Relative band intensities were detected by densitometry using Quantity One 1-D analysis software (version 4.6.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

DPCs were lysed and total protein was extracted. Protein concentrations were quantified by the Bicinchoninic Acid Protein Assay (Thermo Fisher Scientific). About 50 μg of extracted protein samples were loaded into each well, separated with 10% SDS-PAGE, then the protein was transferred onto a PVDF membrane. We used 5% non-fat milk in TBST containing Tween-20 to block the membranes at 37°C for 2 h and then incubated them overnight at 4°C supplemented with primary antibodies against BMP2 (1: 200, sc-137087, Santa Cruz Biotechnology, CA, USA), SMAD5 (1: 200, sc-101151, Santa Cruz Biotechnology), p-Smad (1: 1000, mAb #13820, Cell Signaling Technology, Danvers, MA, USA), RUNX2(1: 200, sc-390715, Santa Cruz Biotechnology), DMP-1 (1: 200, sc-73633, Santa Cruz Biotechnology), DSPP (1: 200, sc-73632, Santa Cruz Biotechnology), and ALP (1: 200, sc-373737, Santa Cruz Biotechnology). Membranes were washed with TBST, incubated with IRDye800 conjugated secondary antibody at 37°C for 60 min, and scanned with the Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, USA). Data were normalized to β-actin (1: 1000, mAb #3700, Cell Signaling Technology) levels and analyzed with Image-Pro Plus software, version 7.0 (Rockville, MD, USA).