Immunoblotting

The cells was lysed in ice-cold RIPA lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS) with protease and phosphatase inhibitor cocktail (catalog #78443, Thermo Scientific) for 30 min on ice. Supernatant was colleceted after centrifugation at 14,000 rpm for 30 min at 4°C. Equal amounts of protein were resolved by SDS-polyacrylamide gel electrophoresis and electroblotted onto equilibrated nitrocellulose membrane. After blocking with 5% milk, the membranes were immunodetected for target proteins. Antibodies against MRP4 (1:100, Abcam, ab15602); phospho-CREB (1:1000, Cell Signaling, 9198), β-tubulin (1:2000, Santa Cruz, sc-9104) and β-actin (1:5000, Sigma, A1978). The protein bands were detected with HRP-conjugated antibodies and visualized by the enhanced chemiluminescence (ECL) assay (GE Healthcare) following manufacturer’s instructions. Signals were quantified by Imagine J software and defined as the ratio of target protein relative to internal loading control.