Quantification of MRP4 Protein Levels

The levels of MRP4 protein were quantified as described in our previous report^{57 (link)}. BMCs prepared from mouse femora were homogenized in lysis buffer containing appropriate protease inhibitors (100 μM phenylmethanesulfonyl fluoride, 2 μg/ml of leupeptin, and 2 μg/ml of aprotinin) and then centrifugated at 4 °C for 10 min at 12,000 × g. The supernatants were denatured at 99 °C for 5 min with 1% SDS and 5% 2-mercaptethanol. Denatured samples containing 20 µg of protein were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were reacted with antibodies against MRP4 (1:5,000; ab15602; Abcam, Cambridge, UK) and β-ACTIN (1:2000; sc-1616; Santa Cruz Biotechnology, Texas, USA). Specific antigen–antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies (1:10,000; sc-2032; Santa Cruz Biotechnology) and ImmunoStar LD (Wako chemicals).