Flag m2 conjugated agarose beads

Cells were harvested and lysed by sonication with a Diagenode Bioruptor UCD-200 in cell lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 1% protease inhibitor cocktails; Sigma-Aldrich) for co-immunoprecipitation analysis (Co-IP). Cell lysates were centrifuged, and the supernatant was incubated with the appropriate antibodies and Protein G Plus/Protein A agarose beads (Calbiochem) or FLAG M2-conjugated agarose beads (Sigma-Aldrich) at 4°C overnight. The beads were washed six times with cell lysis buffer, and the precipitated proteins were further analyzed. For western blotting, equal amounts of protein (80–100 µg) from cell lysates were denatured in sample buffer (Thermo Fisher Scientific), subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad), and transferred to nitrocellulose membranes (Thermo Fisher Scientific). The membranes were blocked in 1 × tris buffered saline Tween (TBST) with 5% milk and immunoblotted with the appropriate primary antibodies at 4°C overnight, followed by incubation at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies. Bands were developed using an enhanced chemiluminescence (ECL) kit (Millipore, Billerica, MA, USA).