Protein Extraction and Western Blotting for Analyzing Cellular Responses to PEMF

The procedures of protein extraction and western blotting were described as in details in our previous study^{29 (link)}. In brief, cells in the PEMF group were exposed to PEMF at 37 °C for 3 days (2 h/day), and then protein lysates were prepared were prepared using RIPA buffer supplemented with 1 mM PMSF on ice. After quantification, protein extracts in equal amounts were loaded on a 10% Tris–glycine SDS-PAGE gel, and then electrotransferred to PVDF membranes. The blots were blocked in 5% bovine serum albumin (BSA) for 1 h, followed by incubation with primary antibodies against OCN (Abcam, Cambridge, MA), Runx2 (Biorbyt Ltd., Cambridge, UK), Wnt3a (Novus Biologicals, Littleton, CO), p-GSK-3β (Abcam), β-catenin (EMD Millipore, Billerica, MA) and β-tubulin (Bioworld technology, Inc., Louis Park, MN) in TBST containing 5% BSA overnight at 4 °C. β-tubulin was employed as the protein loading control. After incubation with a 1:3000 dilution of HRP-conjugated secondary antibody for 1 h at room temperature, the membranes were visualized using the ECL chemiluminescence system (GE ImageQuant 350, GE Healthcare). The Quantity One Software (Bio-Rad) was used to quantify the densities of the bands.