GST Pulldown Assay for Protein Interactions

GST pulldown assays with in vitro–translated [³⁵S]-labeled proteins were done as described previously (Kimura et al., 2015 (link); Chauhan et al., 2016 (link)). All GST-tagged recombinant proteins were expressed in Escherichia coli BL21(DE3) and/or SoluBL21 (Amsbio). GST fusion proteins were purified on Glutathione Sepharose 4 Fast-Flow beads (GE Healthcare). [³⁵S]-labeled Myc-tagged proteins were cotranscribed/translated in vitro using the TnT T7–coupled reticulocyte lysate system (Promega). The in vitro–translated [³⁵S]-labeled Myc-tagged proteins were then incubated with GST-tagged proteins in 250 µl of NETN-E buffer (50 mm Tris, pH 8.0, 100 mm NaCl, 6 mm EDTA, 6 mm EGTA, 0.5% NP-40, and 1 mm dithiothreitol supplemented with cOmplete mini EDTA-free protease inhibitor cocktail [Roche]) for 2 h at 4°C and then washed five times with 1 ml of NETN-E buffer, boiled with 2× SDS gel loading buffer, and subjected to SDS-PAGE. The separated proteins were then transferred to polyvinylidene difluoride membranes using the Trans-Blot Turbo Transfer system (Bio-Rad Laboratories). The GST-tagged proteins were detected by staining with Ponceau S, whereas the radiolabeled proteins were detected in a PharosFX imager (Bio-Rad Laboratories). ​