Ab133612

Western blot analysis was employed as a semiquantitative assay of the expression levels of ALP and OCN. The total proteins were extracted by using a radioactive immune precipitation assay (RIPA) (Solarbio) lysate from rGMSCs which had been incubated for 7 days and 14 days in different groups. To assure concentrations of protein from each sample, the bicinchoninic acid (BCA) method (Thermo) was used. Samples were then submerged in boiling water for 10 minutes. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Subsequently, membranes were blocked with Tris Buffer Solution Tween (TBST) (5% nonfat milk, 0.1% Tween-20) and then incubated at 4°C overnight with the primary antibodies containing ALP (diluted to 1 : 1000; Abgent, AP13552a, USA), OCN (diluted to 1 : 5000; Abcam, ab133612, USA), and β-actin (diluted to 1 : 1000; Proteintech, 60008-1-Ig, USA). After full washing with TBST, samples were incubated with corresponding secondary antibodies (HRP-conjugated goat anti-rabbit IgG, A0208; diluted to 1 : 1000; Beyotime, China) at room temperature for 2 h. Immunoreaction was visualized by an enhanced chemiluminescence assay (ECL, PerkinElmer, USA), and the grey values of protein bands were quantified by using ImageJ.

The total protein content was extracted using the RIPA lysis buffer (Beyotime) with protease inhibitors, followed by protein concentration quantitation using a BCA kit (20201ES76, Yeason, Shanghai, China). Protein was separated by PAGE and then transferred onto PVDF membrane by a wet transfer. Following blocking with 5% BSA, membranes were probed with the primary antibodies against SOX5 (ab94396, Abcam, 1:1000), EZH2 (ab186006, Abcam, 1:2000), osteocalcin (OCN; ab133612, Abcam, 1:1000), Runx2 (ab236639, Abcam, 1:1000), Collagen I (ab34710, Abcam, 1:2000), GAPDH (ab8245, Abcam, 1:3000; internal reference) overnight at 4 °C. The membrane was then re-probed with diluted secondary antibodies against HRP-labeled goat anti-rabbit IgG (ab6721, Abcam) or goat anti-mouse IgG (ab6789, Abcam) for 1 h at room temperature. Following visualization in chemiluminescence reagent, protein quantitative analysis was conducted by the ImageJ software.

Osteogenic differentiation-related proteins including osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), and collagen type I alpha1 (COL-1) were measured in PDLSCs. Total protein was extracted from PDLSCs by lysis in RIPA buffer. Protein concentrations were determined with the BCA kit (Sigma-Aldrich, NJ, USA). Additionally, proteins were isolated using 12% SDS-PAGE gel and were moved onto PVDF membranes (Bio-rad, CA, USA). The membranes were blocked with 5% milk for 2 h and then incubated with primary antibodies for one night at 4°C. Immune complexes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (Abcam, CA, USA). Finally, protein expressions were captured using the Western-Light Chemiluminescent Detection System (Peiqing, Shanghai, China). All experiments were repeated three times. Primary antibodies were purchased from Abcam (CA, USA): anti-OCN (1/1,000, ab133612), anti-RUNX2 (1/1,000, ab236639), anti-COL-1 (1/1,000, ab34710), and GADPH (1/1,000, ab8245).

The cells were treated with RIPA cleavage buffer containing phosphatase and protease inhibitor (Sigma-Aldrich, USA, #89900). The protein concentration was quantified by the BCA protein detection kit (ThermoFisher, USA, #23225). The protein was separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to the PVDF membrane. The imprinted membrane was blocked by TBST containing 5% BSA for 1 h. The first antibody against BMP2 (Abcam, UK, #ab214821), P-SMAD1/5/8 (Santa Cruz, USA, #sc-12353P), RUNX2 (Abcam, UK, #ab236639), OCN (Abcam, UK, #ab133612), PPARγ(Abcam, UK, #ab178860), and β-actin (Abcam, UK, #ab8226) was incubated at 4°C for 14 h, and then the membrane was washed and incubated with goat anti-rabbit (ThermoFisher, USA, #A21076) or goat anti-mouse (ThermoFisher, USA, #A21094) secondary antibody at room temperature for 1 h. The protein was observed by the ECL Western blotting imaging system (ThermoFisher, USA, #32209). The Image Lab software (Bio-Rad, USA) was used to quantify the gray value of protein bands.

Cells were lysed with RIPA lysis solution (Beyotime) to obtain protein samples. After measuring protein concentration with BCA kit (Beyotime), the protein was added into a corresponding volume of loading buffer (Beyotime) and fully mixed. The mixture was heated in boiling water for 5 min to denature proteins. Then, proteins were transferred to PVDF membrane, followed by 1 h blocking using 5% nonfat dry milk. Primary antibodies (GAPDH (5174S, 1 : 1000, Cell Signaling, Boston, USA), RUNX2 (# 12556S, 1 : 1000, Cell Signaling, Boston, USA), OPN (ab8448, 1 : 1000, Abcam, Boston, USA), OCN (ab133612, 1 : 1000, Abcam, Boston, USA), and EZH2 (# 5246S, 1 : 1000, Cell Signaling, Boston, USA)) were added into the membrane and incubated overnight at 4°C. The next day, the membrane was rinsed 3 times prior to 1 h incubation with secondary antibodies (horseradish peroxidase-labeled IgG, 1 : 5000, Beijing CoWin Biosciences Co., Ltd., China, Beijing) at room temperature. After the developer was dropped on the membrane, the detection was performed by chemiluminescence imaging system (Bio-Rad).

L-glutathione reduced (GSH), chloroauric acid (HAuCl₄·3H₂O), b-1-ethyl-3-(3-dimethylamonipropyl) carbodiimide (EDC), sulfo-N-hydroxysuccinimide (s-NHS), bovine serum albumin (BSA), phosphate buffered saline (PBS), PBS-Tween 20, and potassium hexacyanoferrate (III) (K₃[Fe(CN)₆]) were purchased from Sigma-Aldrich (Diegem, Belgium). PBS solution (10 mM, NaCl 0.138 M, KCl 0.0027 M, pH 7.4), and PBS-Tween 20 were prepared by dissolving one package in 1000 mL of de-ionized (DI) water. PBS was used to prepare the antibody (10 µg/mL) and antigen solutions. All other solvents and chemicals were of analytical grade.
For functionalization of the electrodes, monoclonal osteocalcin antibody (ab133612) and full-length osteocalcin protein (ab152231) was obtained from Abcam Co. (Cambridge, UK). Anti-collagen type I antibody (MAB1340) and human collagen type I (CC050) were obtained from Merck Chemicals (Overijse, Belgium).
Double-coated polyester diagnostic tape 9965 (3M Medical Specialties, St. Paul, MN, USA) was used. It consisted of a 0.05 mm opaque white polyester film coated on both sides with a 0.02 mm neutral pressure sensitive acrylate adhesive and supplied between two clear, 0.05 mm silicone-coated polyester release liners.