Figure 6.

ARRDC3 expression is required for activated PAR1 lysosomal trafficking. A, MDA-MB-231 HA-ARRDC3 pSLIK cells incubated with 100 nm LysoTracker were fixed, processed, immunostained for LAMP1, and imaged by confocal microscopy. Images are representative of many cells examined in three independent experiments. Scale bars, 10 μm. Line-scan analysis of the white dotted line region is plotted as the fraction of maximum pixel intensity versus pixel number (distance) and demonstrates that LysoTracker (red) accumulates in the lumen of LAMP1-positive lysosomes (green). MDA-MB-231 HA-ARRDC3 pSLIK cells treated without (B) or with (C) 1 μg/ml DOX for 48 h were incubated with 2 mm leupeptin and 100 nm LysoTracker at 37 °C. Cells were then incubated with anti-PAR1 antibody to label the surface cohort, stimulated with 100 μm TFLLRN, fixed, processed, and imaged by confocal microscopy. Images are representative of many cells examined in three independent experiments. Scale bars, 10 μm. Line-scan analysis of the white dotted line region is plotted as described above and indicates that activated PAR1 (green) accumulates in the lumen of LAMP1-positive lysosomes (red) in the presence of ARRDC3 (blue) (E) but not in lysosomes in the absence of ARRDC3 expression (D).