Localization of endogenous SIMPLE to organelles enriched in PI4P and recycling endosomes in IMS32 cells.

<p>(A) Subcellular fractionation of IMS32 cell lysates was conducted by discontinuous sucrose density-gradient centrifugation and collected into 12 fractions. Equal volumes from each fraction were subjected to SDS-PAGE followed by immunoblot analysis. Previous studies suggested fraction 5 (Red) was rich in PI4P [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199829#pone.0199829.ref024" target="_blank">24</a>]. (B) Co-localization of immunofluorescence within IMS32 cells imaged using confocal microscopy. Left-side panels show endogenous SIMPLE or transiently expressed EGFP-Rab11 (Green). Middle panels show endogenous Rab11 or PI4P in the same cells labeled with specific antibodies (Purple). Merged images are shown on the right with DAPI-stained nuclei (blue). Scale bar, 20 μm. (C) IMS32 cells were transiently transfected with EGFP-tagged full length SIMPLE or SIMPLE (1–90). Twenty-four hours post-transfection the cells were immunostained with anti-PI4P antibody. Left-side panels show EGFP-tagged full length SIMPLE or SIMMPLE (1–90) (Green). Middle panels show PI4P in the same cells labeled with specific antibodies (Red). Merged images are shown on the right with DAPI-stained nuclei (blue). Scale bar, 20 μm.