GTP-agarose pulldown assay

The GTP-agarose pulldown assay was performed as described (25). Briefly, mouse hearts were homogenized using Physcotron (Microtec, Japan) in ice-cold GTP-binding buffer [50 mM Hepes (pH 7.4), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, and 1% protease inhibitor cocktail]. NRCMs were washed with ice-cold PBS, collected in GTP-binding buffer, and homogenized by sonication for 5 s (Qsonica, WakenBtech, Japan). The lysate was centrifuged (16,000g for 10 min at 4°C), and an aliquot of the supernatant (100 μg of protein) was incubated with 50 μl of GTP-agarose beads (equilibrated in GTP-binding buffer) for 45 min at room temperature. The beads were centrifuged (1000g for 1 min at 4°C) and washed twice with GTP-binding buffer. The GTP-bound proteins were eluted by 2× Laemmli buffer with 2-mercaptoethanol and subjected to SDS–polyacrylamide gel electrophoresis (SDS-PAGE).