Protein Extraction and Western Blot Analysis

Proteins extraction and western blot analysis were carried out as described previously [7 (link),9 (link)]. Briefly, cellular proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After blocking the nonspecific binding with 5% nonfat dry milk or 3% BSA for 1 h, the membranes were reacted with the first antibodies overnight at 4°C, which was followed by incubation with the second horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibody. The bands in the membranes were visualized using Chemi-Lumi One L (Nacalai Tesque, Kyoto, Japan) and captured with a Fujifilm luminescent image LAS-1000 analyzer (Fujifilm, Tokyo, Japan).