2.6. Western Blot Analysis

The ER-mediated effects of quercetin on BMP signaling pathway activation and maker protein expression were investigated by western blotting analysis. BMSCs were treated with 2.5 μM quercetin or vehicle control in the absence or presence of 1 μM ICI182780 for 7 days, after which the expression of BMP2, Smad1, p-Smad1, Smad4, RUNX2, OSX, and OPN protein was assessed. For western blotting analysis, the cells were lysed in cold Nonidet P-40 (NP-40) lysis buffer (pH 7.6) containing 50 mM Tris–HCl, 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin for 15 min at 4°C. The cell lysates were collected by scraping and then centrifuged at 14,000g for 10 min. Protein concentrations were assessed using BCA protein assay reagent (Jiangsu Keygen Biotech Corp., Nanjing, China). Twenty micrograms of whole-cell lysates was electrophoresed on an SDS-polyacrylamide gel before being transferred to a PVDF membrane (Jiangsu Keygen Biotech Corp., Nanjing, China), which was blocked in PBS containing 6% low-fat milk and 0.1% Tween 20 (PBST). The blots were then incubated with RUNX2-, BMP2-, Smad1-, p-Smad1-, Smad4-, OSX-, or OPN-antibodies (Jiangsu Keygen Biotech Corp., Nanjing, China), washed twice with PBST, and probed with horseradish peroxidase-conjugated goat anti-mouse secondary antibodies. The protein bands were visualized using a G:BOXChemiXR5 System (Syngene, Cambridge, UK), and the western blotting results were quantified using Gel-Pro32 (Media Cybernetics).