ARF6 localizes to plasma membrane and endosomes and it is crucial to IL-15 secretion after TNF treatment.

<p>HeLa cells were transfected with pCDNA3.1 (control), WT-ARF6, or DN-ARF6 plasmids 48 h before the assay. (A) In control cells and those overexpressing WT-ARF6, TNF treatment for 4 h increased IL-15 release. The cells overexpressing DN-ARF6 did not show a response. In none of the groups did PBS vehicle treatment increase IL-15 release. Data are representative of three independent experiments. *: p<0.05 compared with the control. (B) The overexpression of WT-ARF6 and/or TNF treatment seemed to increase the expression of IL-15 and IL-15Rα, shown by WB of whole cell lysates. (C) The potential interaction of ARF6 with IL-15 was shown by ICC. The left panel is ARF6 immunoreactivity (red, stained with anti-ARF6 in control cells and anti-HA in cells overexpressing ARF6) and the right panel is IL-15 immunoreactivity (green). In control cells, endogenous ARF6 showed a diffuse vesicular pattern of cytoplasmic distribution. In cells overexpressing WT-ARF6 that increased overall expression as well as cell surface distribution of ARF6, IL-15 signal was also increased and showed cell surface distribution co-localizing with WT-ARF6. In cells overexpressing DN-ARF6, the small increase of ARF6 signal remained cytoplasmic, and did not show co-localization with IL-15. Bar: 10 μm. D) Intracellular distribution of IL-15 Rα (green) did not show co-localization with endogenous ARF6 in control cells (stained with an ARF6 Ab, red) or in those overexpressing WT-ARF6 (middle panel) or DN-ARF6 (lower panel, HA Ab staining, red). Bar: 10 μm.