Figure 5: PACS-2 diverts endocytosed ADAM17 away from degradative pathways.

(a) MDA-MB-231 cells were surface labelled using cleavable biotin. Labelled cell-surface proteins were allowed to internalize for 30 min and remaining biotinylated proteins on the cell surface were removed (Intern.). Internalized proteins were then allowed to recycle for 30 min, after which recycled protein was stripped from the cell surface (Recyc.). The amount of cell-surface ADAM17 was first normalized to input actin. The percentage internalization was calculated relative to total cell-surface ADAM17 levels (Total). Recycling was determined by first subtracting the amount of biotinylated protein left after recycling from the amount of internalized protein, and then calculated as a percentage of internalized protein. To verify proper stripping of cell-surface proteins, a dish kept at 4 °C was processed in parallel (Strip). Data were compiled from five individual experiments. (b) MDA-MB-231 cells were surface labelled using non-cleavable biotin, lysed at time zero, or incubated for 4 h at 37 °C to allow internalization and degradation of surface proteins. Percentage degradation was calculated by normalizing the amount of cell-surface ADAM17 to input actin, and dividing the amount remaining after 4 h with the amount present at time zero. Data were compiled from three individual experiments. The images shown are derived from the same blot and same exposure, but have been cropped for clarity (indicated by a separation line). On blots, # denotes a nonspecific band. Graphs show mean values±s.e.m. Data were analysed by unpaired two-tailed Student’s t-test. *P<0.05.