RASSF4 interacts with ARF6 and regulates its localization and activation. (A, left) Confocal images of HeLa cells expressing ARF6-mCherry, treated with either siRASSF4 or siControl. Bar, 5 µm. (Right) The percentage of cells with internal ARF6-mCherry aggregates was calculated from >400 cells across two independent experiments. Means ± SEM are shown. ***, P < 0.001. (B) ARF6 activity was measured in HeLa cells expressing ARF6-mCherry, treated with either siControl or siRASSF4. Anti-ARF6 and anti-mCherry antibodies were used to detect total ARF6-GTP and total ARF6, respectively, by immunoblotting. Total ARF6 was derived from 10% of input lysates. (C) Lysates from HeLa cells transfected with the indicated constructs were subjected to anti-mCherry immunoprecipitation (IP) and subsequent immunoblotting (IB) with anti-GFP and anti-mCherry. (D, left) Lysates of HeLa cells transfected with RASSF4-YFP and the indicated ARF6 construct were subjected to anti-GFP immunoprecipitation and subsequent immunoblotting with anti-GFP and anti-mCherry. (Right) Relative intensities (IP/Input) of ARF6 and its variants determined by ImageJ. (B–D) The unit of molecular weight shown next to immunoblots is kilodaltons. Similar results were obtained in more than three independent experiments. (E) Intracellular Ca2+ levels determined by analysis of Fura-2 fluorescence ratios in HeLa cells treated with ARF6-N122I or a control vector, stimulated with 1 µM TG and 100 µM histamine (His). 2 mM Ca2+ was added 6 min after stimulation. Shown are mean Fura-2 ratios ± SEM derived from >350 cells for each condition across two independent experiments. (F) Model of PM PI(4,5)P2 regulation by RASSF4 through the ARF6-PIP5K pathway.
