Pvdf membranes

The protein expression of odontogenic-related genes, including dentin sialophosphoprotein (DSPP), dentin matrix acidic phosphoprotein 1 (DMP-1), alkaline phosphatase (ALP), and runt-related transcription factor 2 (Runx2), was detected by Western blot analysis. The DPSCs were appropriately cultured in an odontogenic medium containing either 20 µg/mL CM-EV or OM-EV for 7 and 14 days as explained above. Cells were then collected and lysed in RIPA buffer (KeyGen BioTECH, Nanjing, China) containing a 1% protease inhibitor cocktail (CWBIO, China). The protein concentration was determined by employing the BCA assay kit (CWBIO). The proteins of each group were separated by 10% SDS-PAGE (GenSpirt, China) and transferred to PVDF membranes (Millipore, USA). After blocking with 5% fat-free milk, the PVDF membranes were subsequently incubated overnight at 4 °C with primary antibodies anti-DSPP (1:500, Novus, USA), anti-DMP-1 (1: 500, Bioss), anti-Runx2 (1:1000, Novus), anti-ALP (1:1000, Novus), anti-GAPDH (1:1500, Novus), followed by incubation with secondary antibody (1:5000, GNI, Japan). An advanced chemiluminescence detection system (Millipore) was utilized to detect immunoblots with an imaging system (ChemiDoc, Bio-Rad, China). The bands’ intensities were suitably measured via ImageJ and normalized to GAPDH.

To assess for SETX depletion, western blot analysis was performed with NuPAGE Bis–Tris 4–12% gels and reagents (Invitrogen) according to the manufacturer’s instructions. Briefly, cells were lysed in NuPage sample buffer with reducing agent (Invitrogen) and resolved proteins were transfered onto PVDF membranes (Invitrogen). PVDF membranes were then saturated 1 h in 5% nonfat dry milk with TBS and 0.5% Tween 20 and incubated overnight with the following primary antibodies: anti-SETX (Novus Biologicals, NB100-57542, 1:500) and anti-alpha-tubulin (Sigma-Aldrich, DM1A, 1: 100,000). Horseradish peroxidase-coupled secondary antibodies were from Sigma (anti-mouse, A2554, 1: 10,000; anti-rabbit, A0545, 1: 10,000), and the chemiluminescence Lumilight reagent was from Roche Diagnostic. To analyze I-SceI expression, total cell lysates were prepared 24 h after I-SceI plasmid transfection by direct resuspension of cells in Laemmli buffer and sonication. Cell extracts were separated on 10% SDS PAGE and proteins were transferred on nitrocellulose membrane. Primary antibodies were anti-myc (9E10, Roche) and anti-alpha-tubulin (Sigma-Aldrich). Signals were analyzed by autoradiography (for SETX expression) or using a ChemiDoc touch device (BioRad) for I-SceI expression.

The L3–L5 DRGs were rapidly homogenized and lysed in RIPA lysis buffer on ice. The protein concentration was determined with a BCA protein assay kit (Thermo Scientific, United States). The protein was electrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel and then transferred to polyvinyl difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, United States). The PVDF membranes were incubated overnight at 4°C with primary rabbit-anti-CCR2 (1:500, Novus Biologicals, NBP1-48337), rabbit-anti-HCN2 (1:300, Almone Labs, APC-030) or mouse-anti-β-actin (1:5000, Abcam, ab8227) and were incubated with the secondary antibody Donkey anti-Rabbit (1:4000, bioss, bs-0295D) or anti-mouse IgG (1:4000, Cell Signaling Technology, #7056) at ambient temperature for 2 h. An appropriate amount of ZETA luminescent solution was taken, and the corresponding images were saved using a Bio-Rad luminometer for luminescence the protein bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, United States).