Coimmunoprecipitation and Immunoblot Analyses

Coimmunoprecipitation and immunoblot analyses were performed as previously described.^{46 (link)} In brief, cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40) containing protease and phosphatase inhibitor cocktail (Selleck, Houston, Texas, USA). For coimmunoprecipitation, lysates were incubated overnight with ANTI-FLAG^® M2 Affinity Gel (Sigma-Aldrich), EZview™ Red Anti-HA Affinity Gel (Millipore, Billerica, MA, USA), or Pierce Glutathione Agarose (Thermo Scientific, Rockford, IL, USA), then proteins were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking in TBST containing 5% BSA, the blots were probed with primary antibodies. Determination of the band intensities were performed with ChemiDoc XRS⁺ Molecular Imager software (Bio-Rad, Philadelphia, PA, USA).