FIGURE 5.

ARAP2 controls integrin traffic from APPL to EEA1 endosomes. A–D, colocalization of integrin with APPL or with EEA1 was examined. siRNA-treated HeLa and MDA-MB-231 cells expressing ARAP2 (FLAG-WT, FLAG-RK) and EGFP bicistronically or EGFP alone were costained for inactive β1 integrin (MAB13) and APPL or EEA1. Transfected cells were identified by GFP in the nucleus (shown in blue and in white boxes). Boxed areas are shown in higher magnification, with arrows indicating structures where colocalization was detected. Representative images (A and B) and quantification of colocalization between integrin and APPL or EEA1 (C and D) are shown (n = 20∼41) from at least three experiments. Scale bars = 10 μm. ns, not significant. *, p < 0.05. E, ARAP2 affects the association of active integrin with organelles in the endocytic pathway. siRNA-treated HeLa cells were coimmunostained for active β1 integrin (9EG7) and markers for different organelles: APPL, EEA1, and LAMP1. Alternatively, siRNA-treated cells were transfected with plasmids for expressing different Rab proteins and stained for active β1 integrin (9EG7 or 12G10). *, p < 0.05. F–H, ARAP2 affects the cellular distribution of APPL endosomes. siRNA-treated HeLa and MDA-MB-231 cells were stained for APPL. The distribution of APPL was scored blindly as peripheral or non-peripheral for 40∼80 cells from each group in each experiment. Representative images from HeLa cells (F) and quantification presented as the mean ± S.E. from three experiments (G and H) are shown. Scale bars = 10 μm. *, p < 0.05; **, p < 0.005.