GST pull down assay

GST pull down of GST–Rab11a with Myo5b–GTD and FIP2–RBD was performed as follows. One hundred microliters of 0.5 μM GST–Rab11a, 0–1.0 μM Flag-Myo5b-GTD and 0–1.0 μM Flag-FIP2-RBD in Buffer-B [20 mM MOPS-KOH (pH 7.0), 1 mM MgCl₂, 1 mM EGTA, 1 mM DTT and protease inhibitor cocktail (Roch)] containing 100 mM NaCl and 3 μM GTPγS were mixed with 20 μl of GSH-Sepharose and incubated with rotation at 4°C for 1 h. The bead was collected by brief centrifugation and washed with 100 μl of Buffer-B containing 50 mM NaCl and 1 μM GTPγS for five times. The bound proteins were eluted with 50 μl of 20 mM GSH in Tris-HCl (pH 8.0), 100 mM NaCl and 1mM DTT. The eluted proteins were separated by SDS−PAGE (4–20%) and visualized Western blot.

GST pull down of GST–Rab11a with Myo5b–Tail and FIP2 or FIP2–RBD was performed as follows. One hundred microliters of 0.5 μM GST–Rab11a, 0–0.5 μM Flag-Myo5b-Tail and 0–1.0 μM Flag-FIP2 or Flag-FIP2-RBD in Buffer-B containing 100 mM NaCl and 3 μM GTPγS were mixed with 20 μl of GSH-Sepharose and incubated with rotation at 4°C for 1 h. The bead was collected by brief centrifugation and washed with 100 μl of Buffer-B containing 50 mM NaCl and 1 μM GTPγS for five times. The bound proteins were eluted with 50 μl of 20 mM GSH in Tris-HCl (pH 8.0), 100 mM NaCl and 1 mM DTT. The eluted proteins were separated by SDS−PAGE (4–20%) and visualized by Coomassie Brilliant Blue staining.