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. 2015 Oct 27;5:14919. doi: 10.1038/srep14919

Figure 1. Dyn2 activates Arf6 in a manner dependent on its GTPase activity.

Figure 1

(A) HA-tagged wild type of Dyn2 or its GTPase-deficient mutant K44A was coexpressed with Arf6-Flag in HeLa cells. After 24 hr, the active GTP-Arf6 was pulled down with glutathione-Sepharose beads conjugated with glutathione S-transferase (GST)-tagged leucine zipper region II (LZII) (amino acids 398–455) of JNK-interacting protein (JIP), which specifically binds to the active form of Arf6, and immunoblotted with anti-Flag antibody (left panel). Total Arf6 and Dyn2 expressed in the cell were also immunoblotted with anti-Flag and -HA antibodies, respectively. Right panel shows the means ± SEM of the levels of GTP-Arf6 from eight independent experiments. Statistical significance was calculated using Tukey multiple comparison test; **P < 0.01 and N.S., not significant. (B) HeLa cells expressed with wild type of HA-Dyn2 or its K44A mutant and Arf6-Flag were treated with 80 μM dynasore at 37 °C for 30 min, and the levels of GTP-Arf6 and total Arf6 were detected as in (A) (left panel). Right panel shows the means ± SEM of the levels of GTP-Arf6 from three independent experiments. Statistical significance was calculated using Tukey multiple comparison test; *P < 0.05 and N.S., not significant.