Full-length RPH3A and RAB21 were expressed in E. coli BL21(DE3) and purified by affinity purification. Myristoylated ARF6 protein and phosphorylated RPH3A (pRPH3A) protein were prepared as described in (59 (link)) and (16 (link)), respectively. For the pull-down assays, recombinant RAB21 protein or myristoylated ARF6 protein (0.2 mg/mL in 10 μL dissolved in buffer A containing 10 mM phosphate buffer [pH 7.4], 10% glycerol, and 0.2 mM EDTA) was added with 1 μL of 10 mM GDPγS or GTPγS stock (dissolved in 50 mM Tris-HCl [pH 7.8]) for a final concentration of 1 mM guanine nucleotides and incubated on ice for 15 min. Then, RPH3A (0.2 mg/mL in 10 μL dissolved in buffer A) and 180 μL buffer B (10mM phosphate buffer [pH 7.4], 10% glycerol, 1% Triton X-100, 0.1% SDS, 3 mM DTT, protease inhibitor cocktail, 10mM MgCl2, and 0.1mM GTPγS or GDPγS, with 0.1mM CaCl2) were added to RAB21 or ARF6 protein and incubated at 4°C on a rotating platform for overnight. The protein mixture was then added to Ni-NTA agarose beads (GE Healthcare) pre-blocked with 1% BSA and incubated at 4°C on a rotating platform for 2 hr. After the beads were washed six times with buffer B, immune complexes were released from the beads by boiling and analyzed by western blotting.