Ab15602

The cells or tissues were lysed in ice-cold RIPA lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% DOC and 0.1% SDS) with protease and phosphatase inhibitor cocktail (catalog #78443, Thermo Scientific) for 30 min on ice. Supernatant was collected after centrifugation at 13,000 rpm for 30 min. Equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis and electro-blotted onto equilibrated nitrocellulose membrane. After blocking in Tris-buffered saline (TBS) containing 5% non-fat milk, the membranes were immunoblotted with primary antibody of target proteins overnight at 4 ^oC. Antibodies against MRP4 (1:100, Abcam, ab15602); Non-phospho (Active) β-catenin (1:1000, Cell Signaling, 8814), β-catenin (1:1000, Cell Signaling, 9562), p-β-catenin (1:1000, Cell Signaling, 9561), β-tubulin (1:2000, Santa Cruz, sc-9104) and β-actin (1:5000, Sigma, A1978) were used. After three washes in TBS containing 0.1% Tween 20 (TBST), membranes were further incubated with HRP-conjugated antibodies and visualized by the enhanced chemiluminescence assay (GE Healthcare, UK) following manufacturer's instructions. Densitometry of Western blots was performed by Image J software.

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).