Horseradish peroxidase conjugated anti rabbit or anti mouse igg antibodies

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).

Cells were lysed in lysis buffer (Thermo Fisher Scientific), and protein levels were measured using the BCA method (Thermo Fisher Scientific). Fifty micrograms of proteins was separated by 10–15% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (PVDF, Thermo Fisher Scientific) using a semi-dry blotter. The membrane was blocked with 5% BSA for 1 h and then incubated with specific primary antibodies: anti-TGF-β1, anti-α-SMA, anti-cTnT, anti-p-Smad2/3, anti-Smad2/3, or anti-actin (antibodies were all purchased from Abcam, MA, USA). Following overnight incubation, the membranes were washed with 1% TBST three times (15 min each) and incubated in horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (Abcam) at room temperature for 50 min. Following incubation, the membranes were washed with 1% TBST three times (15 min each). The protein-antibody complexes were detected using the chemiluminescent reagent WesternBright ECL detection kit (Thermo Fisher Scientific). Relative protein levels were determined in comparison with β-actin.