4.3. Western Blot Analysis

Briefly, SNU484 and SNU638 cells were seeded and plated to attach for 24 h. UA was added to cell cultures at 0, 10, 25, or 50 µM concentrations and incubated for 72 h. Subsequently, the samples were collected and adjourned in a lysis buffer (Intron Biotechnology, Korea). Cells were extracted and incubated on ice for 10 min. The samples were then centrifuged at 13,200 rpm for 20 min. at 4 °C and the protein concentration was measured (BSA protein assay kit, Pierce Biotechnology, Inc., Rockford, IL, USA). The lysate was resolved on a gel and then moved to the membranes (PVDF, Bio-Rad, Hercules, CA, USA). The specific primary antibodies were displayed and the secondary antibodies were probed to the membranes. Protein bands were visualized while using a chemiluminescence kit (Amersham, Arlington Heights, IL, USA). The following antibodies were used: cleaved-PARP, PARP, cleaved-caspase-9, caspase-9, caspase-3, Rassf1, Mst1, Mst2, Mob1, p-Mob1, LATS1, YAP, p-YAP, CTGF, Sav1, E-cadherin, MMP-9, Vimentin, Twist, and GAPDH. All of the antibodies were diluted to 1:1000. Quantification analysis of Western blotting band was performed while using Image J.