Expression of Erythropoietin Receptor (EPOR) in Bone Marrow Mononuclear Cells by Western Blotting

On day 30, 1 × 10⁷ BMMNCs were collected by group and lysed with ultrasonication in RIPA cell lysis buffer (Beyotime Biotechnology, Haimen, China), supplemented with 1 mM PMSF. The lysates were clarified by centrifugation at 12,000 rpm for 15 min at 4°C. After denatured by boiling in loading buffer (Transgen Biotech, China), the protein was loaded and separated on 12% glycine SDS-PAGE gel, and transferred to polyvinylidene difluoride (PVDF) membranes (0.45 μm; Millipore, Bedford, MA). EPOR (CST, Rabbit pAb) and β-actin (CST, Rabbit mAb) were probed with specific primary antibodies and HRP-conjugated secondary anti-rabbit antibodies (CST, anti-rabbit-IgG). The immune-complex on the membrane was visualized using an automatic chemo-luminescence image analysis system (Tanon, Shanghai, China) with HRP substrate luminol reagent and peroxide solution (Millipore).