Cells were lysed and subjected to immunoblotting as described previously46 (link),47 (link). For co-immunoprecipitation, cells were lysed in Co-IP buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM MgCl2; 10% glycerol and 1% NP-40) supplemented with cOmplete EDTA-Free Protease Inhibitor (Sigma-Aldrich) by incubation for 30 min at 4 °C. Cell pellets were removed by centrifugation and the supernatants were precleaned by incubation with rat or mouse IgG and Protein G Sepharose (GE Healthcare Life Sciences, Pittsburgh, PA, USA) for 30 min at 4 °C under gentle rotation. After centrifugation, the supernatants were co-immunoprecipitated by incubation with anti-HA affinity matrix (11815016001, Sigma-Aldrich), anti-DYKDDDDK tag antibody beads (018-22783, Wako Pure Chemical Industries), or anti-c-myc (9E10) antibody (sc-40, Santa Cruz Biotechnology, Santa Cruz, CA, USA) conjugated with Protein G Sepharose at 4 °C under gentle rotation overnight. The beads were washed with Co-IP buffer three times and then boiled in Laemmli sample buffer. The eluates were subjected to immunoblotting.
qRT-PCR was performed using the primers shown in Supplementary TableS1 as described previously48 (link).
qRT-PCR was performed using the primers shown in Supplementary Table
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