First, 1 × 107 cells were washed with ice-cold PBS and lysed in 500 μL CHAPS buffer supplemented with protease inhibitor cocktail for 30 minutes at 4°C. Lysates were centrifuged at 20,000g for 10 minutes at 4°C. Next, 50 μL supernatants were aliquoted as input sample, supplemented with SDS sample buffer, and incubated for 10 minutes at 95°C. GTP-conjugated agarose beads (Axxora, JBS-AC-106S) were washed once with washing buffer to reduce nonspecific binding. Then, 250 μL supernatants were transferred and incubated with 20 μL GTP-conjugated agarose beads with or without 1 mM GTP overnight at 4°C. The beads were then washed 3 times with washing buffer and incubated with 50 μL SDS sample buffer for 10 minutes at 95°C. The IP complex and 10 μg input samples were subjected to Western blot analysis.

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