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. 2020 Jan 10;204(4):1012–1021. doi: 10.4049/jimmunol.1901080

FIGURE 6.

FIGURE 6.

ARF6 is a coincidence-detection code for RPH3A protein. (A) GTP-bound ARF6 augments the interaction of RPH3A with PtdIns4P. Purified recombinant myristoylated ARF6 protein preloaded with GDPγS or GTPγS was incorporated into phosphocholine (PC)/phosphatidylserine (PS) liposome containing PtdIns4P. The liposomes were then incubated with purified recombinant RPH3A protein, followed by density ultracentrifugation. Input and liposome-associated proteins were detected by Western analysis. (B) RPH3A preferentially binds to GTP-bound ARF6. Purified recombinant myristoylated ARF6 protein preloaded with GTPγS or GDPγS was incubated with purified His-tagged recombinant RPH3A protein, followed by nickel-bead pull-down and Western analysis. (C) ARF6 preferentially binds to p-RPH3A. Purified recombinant proteins were used in an His–pull-down assay under the conditions described in the figure. Proteins were then detected by Western blot analysis. (D) ARF6 augments the interaction between the RPH3A and RAB21. Purified GTPγS-bound myristoylated ARF6 and RAB21 were incubated with p-RPH3A and subjected to an His–pull-down assay. The proteins were detected by Western blot analysis.