Luminescent image analyzer amersham imager 600

HCT116 cells treated with DMSO or 5 μM Manz A for 24 h were lysed for 30 min in ice-cold lysis buffer containing protease and phosphatase inhibitor cocktail. The protein concentration was determined by Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). Thirty μg protein was subjected to SDS-PAGE, resolved on a 7.5–15% polyacrylamide gel, and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked in 5% bovine serum albumin (BSA) for 1 h and incubated with the appropriate primary antibody overnight at 4 °C. After three washes with Tris-buffered saline containing 0.05% Tween-20 (TBST), the membrane was incubated with secondary anti-rabbit or anti-mouse IgG antibodies (1:15,000) for 1 h at room temperature. The immunoreaction was visualized using the ECL HRP substrate and detected using a Luminescent Image Analyzer Amersham Imager 600 (GE Healthcare Life Sciences, MA, USA). The band intensity was quantified using ImageJ software.

Cells were lysed in RIPA buffer containing a protease inhibitor mixture and a phosphatase inhibitor cocktail tablet (Roche Diagnostics, Basel, Switzerland). The protein concentration was analyzed by the bicinchoninic acid (BCA) assay kit (T-Pro Biotechnology, New Taipei City, Taiwan). Quantified protein sample (20 μg) was resolved on 7.5–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then proteins were transferred from the gel to polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked in 5% BSA solution for 1 h and incubated with the anti-RAGE (1:1000; Millipore) and PCNA (1:1000; Cell Signaling) antibodies overnight at 4 °C. The next day, the membrane was incubated with secondary anti-rabbit or anti-mouse antibodies (1:10,000; Cell Signaling) for 1 h at room temperature. The membrane was reacted with the Electrochemiluminescence (ECL) reagent and the visual signals were detected using a Luminescent Image Analyzer Amersham Imager 600 (GE Healthcare Life Sciences, MA, USA). The band densities were quantified using the Image-J software program.