Ab41901

CRC tissue samples and cells were washed with ice-cold phosphate-buffered saline (PBS) 3 times and lysed in RIPA buffer supplemented with protease inhibitor. The protein concentration was determined using the bicinchoninic acid method. Equal amounts of proteins (30 µg) were separated by 15% SDS-PAGE and transferred to a PVDF membrane (Immobilon; Millipore Corp., Bedford, MA, USA). After blocking with 5% non-fat milk (diluted with phosphate-buffered saline/Tween-20, TBST) at 37°C for 1 h, the membranes were incubated with primary antibodies: anti-LRH-1 (1:1000, ab41901), anti-matrix metalloproteinase (MMP)2 (1:1,000, ab80737), anti-MMP9 (1:1,000, ab119906), anti-β-catenin (1:500, ab92514), cyclin D1 (1:1,000, ab134175), c-Myc (1:500, ab51156) and GAPDH (1:1,000, ab8245; Abcam, Cambridge, UK) at 4°C overnight. After rinsing with TBST, they were incubated with horseradish peroxidase-conjugated goat anti-mouse or rabbit immunoglobulin G antibodies (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Protein bands were detected using an enhanced chemiluminescence reagent (Pierce Biotechnology, Inc., Rockford, IL, USA). Band intensity was quantified using Image software (Media Cybernetics, Inc., Rockville, MD, USA).