Ab15602

Uterus tissues were harvested and fixed by immersion in 4% paraformaldehyde overnight at 4 ^oC. After three times washed in PBS, uterine tissues were cryoprotected in 30% sucrose in PBS at 4 ^oC for 24 h, mounted in OCT embedding media (Tissue-Tek, 4583, Sakura), and cryo-sectioned into 5 μm sections. Sections were rehydrated in PBS for 5 mins and microwaved in citrate buffer (pH 6.0) for 20 mins to retrieve antigens. After cooled down to room temperature and treated with 1% SDS in PBS for 4 mins, sections were blocked with 1% bovine serum albumin in PBS for 15 mins, incubated with primary antibody (MRP4, 1:20, Abcam, ab15602) and/or β-catenin (1:100, Cell Signaling, 2677) overnight at 4 ^oC and subsequently fluorochrome-conjugated secondary antibody (invitrogen) for 1 h at room temperature. DAPI was used to stain cell nuclei. Images were acquired with a confocal microscope (Zeiss, Germany).

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.