ATPase Assay
RNF213 proteins fused with EGFP were expressed in HEK293 cells, and cells were extracted with RIPA buffer. The cell extract was clarified by high-speed centrifugation and used for immunoprecipitation with anti-GFP agarose (MBL Japan, Nagoya, Japan). Immunoprecipitants were washed with RIPA buffer twice and with RIPA buffer without SDS once, and finally equilibrated with 0.5× kinase buffer (10 mmol/L of Tris-HCl [pH 7.6], 100 mmol/L of KCl, 5 mmol/L of MgCl2, and 0.025% TritonX-100). Immunoprecipitants were resuspended into 50 μL of kinase buffer, and half of this volume was subjected to SDS-PAGE and stained with GelCode staining (Thermo Scientific). To perform ATPase reactions, the indicated volume of immunoprecipitants was combined with prewarmed 1× kinase buffer with 60 μmol/L of ATP. The 50-μL ATPase reaction proceeded for 30 minutes at room temperature by adding a final concentration of Phosphate Sensor (0.5 μmol/L; Thermo Scientific). The plate was mixed and immediately read on a microplate reader at an excitation of 430 nm and emission of 450 nm.