Quantitative Western Blot Analysis

Western blot analysis was performed according to previous description [19 (link)]. Briefly, the cellular proteins were extracted and loaded onto 6~8% SDS-PAGE. After 120 min electrophoresis with 80 volt, the proteins were transferred onto PVDF membrane. Blocking was performed by incubation with 5% fat-free milk in TBST. The membrane was incubated with primary antibody at 4 °C overnight with 1:1000 dilutions. After washing six times with TBST (for 10 min each), the membrane was further incubated with corresponding HRP-conjugated secondary antibody at room temperature. The bound secondary antibody was visualized using enhanced chemiluminescence ECL (Advansta, CA, USA). β-actin was used as a loading control. Protein level was quantified by ImageJ software and presented as related integrated density (RID).