Arf6 activation assay kit

Basal Arf6 activity level in CentA1 KO mice and their NTG littermates was determined using Cell Biolabs’ Arf6 activation assay kit and following the manufacturer’s instructions. Briefly, the active form of Arf6 from hippocampal lysates was selectively isolated and pulled down with the GGA3 protein-binding domain (GGA3-PBD) attached to agarose beads. The precipitated GTP-bound, and therefore active Arf6, was detected by Western blotting using an anti-Arf6 antibody.

ARF6-GTP pulldown assays were performed according to manufacturer’s instructions using the Arf6 Activation Assay Kit (Cell Biolabs). Briefly, Mel92.1 or Mel202 cells were grown in RPMI-1640/10% FBS to 80% confluency in 10 cm tissue culture dishes and then treated with 1 μM to 5 μM Tris DBA palladium or vehicle (0.1% DMSO in medium) for 3 h at 37° C/5% CO₂. Cells were lysed at 4° C in 1X lysis buffer (Cell Biolabs kit) supplemented with protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). Total cell lysate (0.5 mg) was incubated with GGA3-conjugated agarose beads for 1 hour, and then the beads were washed three times with 1X lysis buffer supplemented with protease and phosphatase inhibitors. Precipitates with beads (ARF6-GTP) and total cell lysates (total ARF6) were analyzed by western blot using 5% nonfat dry milk in PBST as a blocking agent, anti-ARF6 as a primary antibody (diluted 1/1000; Cell Signaling Technology), and a secondary antibody conjugated to horseradish peroxidase (diluted 1/5000; Jackson ImmunoResearch). Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Quantification was performed using scanning densitometry and ImageJ (NIH), and ARF6-GTP levels were normalized to total ARF6 levels.