Analysis for Arf6 activation

Activation of Arf6 was assessed by the Arf6-GTP pulldown assay as described in the previous report by Santy et al.²⁸. Briefly, HepG2 cells were seeded on 3.5 cm dishes at 3 × 10⁵ cells/dish and incubated overnight. After 12 h starvation, cells were stimulated with 10 ng/mL of HGF for 10 min. Cells were harvested in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol, 1 μg/ml of aprotinin, and 1 μg/ml of leupeptin), and lysed at 4 °C for 30 min. The cell extracts were mixed with glutathione S-transferase (GST)-GGA3-conjugated glutathione-Sepharose beads and incubated for 30 min with gentle rotation. The beads were washed three times with the washing buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl₂, 1% NP-40, 10% glycerol, 1 μg/ml of aprotinin, and 1 μg/ml of leupeptin). The Arf6-GTP bound to GST-GGA3 beads was eluted by SDS sample buffer, and detected by Western blotting.