Co-Immunoprecipitation Experiments with Flag-tagged Proteins

Co-IP experiments were performed as previously described (Shyian et al., 2020 (link)) with the following modifications. OD₆₀₀ ~ 0.9 cell cultures were used to prepare 1 ml of lysate. Cells were broken with glass beads in a Precellys Evolution homogenizer. 20 µl of anti-Flag M2 gel (Sigma) per 1 ml of lysate were used. Proteins were eluted by boiling at 95 °C for 10 min in 30 µl of 2 x SDS-PAGE buffer (100 mM Tris pH 8, 4% SDS, 10% glycerol, 0.2% bromophenol blue). Total proteins were isolated as previously described (Kushnirov, 2000 (link)). Proteins were separated on 8% PAGE gels, transferred onto Hybond P 0.22 PVDF membrane (GE Healthcare), stained with Ponceau S (Amresco), and blocked in 5% BSA. Anti-HA-HRP antibodies (clone 3F10, Sigma) at 1:5000, anti-FLAG M2 antibodies (Sigma) at 1:5000, goat anti-mouse IgG-HRP (62–6520, Thermo Fisher Scientific) at 1:5000 and SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) were used for protein detection.
RNA Co-IP experiments were performed as described in Shepelev et al., 2020 (link).