Figure 5.

INPP4A is secreted via unconventional mechanisms into the membrane of extracellular vesicles (EVs) and as a free, nonvesicle-bound form. (A–C) Representative confocal fluorescence images of BEAS-2B cells costained with INPP4A (red) and endosome marker RAB5, or with RABs involved in vesicular exocytosis (RAB27A and RAB27B) (green). Nuclei are stained by DAPI (blue). Scale bar: 5 μm. (D) Bar plot showing mean colocalization rate of INPP4A with the three RABs as calculated by an automated module in Leica LAS AF software. This value indicates the extent of colocalization in percentage. Colocalization rate (%) = area colocalization/area foreground; area foreground = area image − area background. (E) Representative immunoblot of the INPP4A and EV markers tumor susceptibility gene 101 (TSG101) and heat shock protein 70 (HSP70) in the EV fractions isolated by differential ultracentrifugation of the conditioned media of BEAS-2B cells. 10K = 10,000 × g pellet (microvesicles); 100K = 100,000 × g pellet (exosomes). TCL = total cell lysate. INPP4A was also detected in the EV-free fraction, as seen in the trichloroacetic acid (TCA) precipitate of a portion of the supernatant obtained after the 100,000 × g centrifugation (post-100k TCA fraction). Results are expressed as the mean ± SEM.