Immunoblotting Protocol for Total Cell Lysates

All antibodies were purchased from Cell Signaling Technology (Danvers, MA). Total cell lysates were prepared, and immunoblotting was conducted as described previously [11 (link)]. Briefly, cells were cultured until subconfluent, rinsed with phosphate-buffered saline (PBS), lysed in sodium dodecyl sulfate (SDS) sample buffer, and homogenized. The total cell lysate (10 μg) was subjected to SDS polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). After blocking with 5% nonfat dry milk, membranes were incubated with primary antibodies, washed with PBS, and reacted with secondary antibodies (Cell Signaling Technology), and signals visualized using ECL reagent (Clarity, Bio-Rad, Hercules, CA) and film or detected by an ImageQuant Imager (GE Healthcare Bio-Sciences, Tokyo, Japan).