GST Pulldown Assay for IRGM-NLRP3 Interaction

GST pulldown assay was done as described previously (Kimura et al., 2015 (link), Chauhan et al., 2016 (link)). GST-IRGM recombinant protein was expressed in SoluBL21 (Amsbio) and purified on Glutathione Sepharose 4 Fast-Flow beads (GE Healthcare). [35S]-labeled Myc-NLRP3 was cotranscribed/translated using the TnT T7–coupled reticulocyte lysate system (Promega). The in vitro–translated [35S]-labeled Myc-NLRP3 protein was then incubated with GST or GST-IRGM 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 loading buffer, and subjected to SDS-PAGE. Gel was stained with Coomassie Blue and vacuum-dried. The GST-IRGM was detected by staining with Coomassie Blue, whereas the [35S]-labeled Myc-NLRP3 was detected in PharosFX imager (Bio-Rad Laboratories)