Cell Fractionation, Protein Analysis, and Immunoprecipitation

SDS-PAGE, immunoprecipitation, and western blot assays were carried out as described previously (Tanigawa et al. 2013 (link); Pan et al. 2010 (link)). In brief, cell lysates were centrifuged at 500 × g for 5 min at 4 °C to prepare nuclei and cytosol by using the nuclear and cytoplasmic extraction reagents, respectively (NE-PER; 78833; Thermo Fisher Scientific). Cell lysates were fractionated in 10% SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (0.45-μm IPVH00010; Merck) for 1 h at 100 V (fixed) at 10°C using a Mini Trans-Blot transfer system (Bio-Rad Laboratories, Hercules, CA, USA). Blotted proteins were visualized with Ponceau S (Merck, P17170) to examine the amounts of the transferred proteins. PVDF membranes were then incubated with primary and secondary antibodies (Supplementary Table S1). Results were investigated using a ChemiDoc XRS Plus instrument (Bio-Rad). Immunoprecipitation was conducted by protein A/G beads coated by indicated antibodies (Tanigawa et al. 2013 (link); Pan et al. 2010 (link)).