Co-immunoprecipitation Assay Protocol

Co-immunoprecipitation was performed as previously described (Liu et al., 2022 (link)). In brief, cells were lysed in the lysis buffer containing protease and phosphatase inhibitor cocktail (Selleck, Houston, Texas, United States). For co-immunoprecipitation, lysates were incubated overnight with ANTI-FLAG^® M2 Affinity Gel (Sigma-Aldrich). Then, proteins were separated by SDS-PAGE and electro-transferred onto the PVDF membrane. The membrane-containing proteins were blocked for 1 h at room temperature with 5% BSA in PBST, followed by treatment with the indicated primary antibodies overnight at 4°C. Subsequently, blots were incubated with secondary antibody for 1 h at room temperature and visualized via enhanced chemiluminescence reagents (Millipore, Billerica, MA, United States) with ChemiDoc XRS⁺ Molecular Imager software (Bio-Rad, Philadelphia, PA, United States).