Fig. 6. SNX18 is part of an endosomal AP-1- and PACS1-positive budding machinery. (A) Fluorescent micrograph of a HeLa cell treated with BFA and co-stained for endogenous SNX18 and AP-1. Magnification of the boxed area is shown to the right; arrows indicate colocalized puncta. (B) The graph shows the degree of colocalization between SNX18 and AP-1 in untreated and BFA-treated HeLa cells. The bars show the percentage of SNX18-positive puncta that colocalized with AP-1, as means (s.e.m.) from a total of 45 5x5-µm squares placed over 25 randomly selected cells. Student's t-test showed P<0.01 between untreated and treated cells. (C) Lysates from BFA-treated or untreated HeLa cells were immunoprecipitated (IP) with antisera against -adaptin or without primary antibody (Control), and analyzed by immunoblotting (Blot) using antisera against SNX18 and -adaptin. Co-immunoprecipitation increased after BFA-treatment. (D) Epifluorescent micrograph showing a HeLa cell overexpressing myc-tagged SNX18 (green) together with untransfected cells, stained for AP-1 (red) after washout of cytosolic components. For clarity, the cell borders are outlined as broken lines. Note the disappearance of peripheral AP-1 puncta in the SNX18-overexpressing cell. (E) Epifluorescent micrograph showing a HeLa cell stained for endogenous SNX18 and PACS1. Arrows in the magnified merged inset illustrate puncta in which the proteins co-localized. Scale bars: 10 µm.