FIGURE 9.

The guanine nucleotide exchange factor GAPex5 is important for the Rab31-mediated enhancement of ligand-bound EGFR endocytic trafficking. A, A431 EGFP-Rab31 OE cells were transfected with scrambled (Scr) or GAPex5 siRNA. Cells were pulsed with 0.5 μg/ml EGF-TxR and fixed at 30 min for analysis of the size of EGF-TxR puncta (red) in cells that do (green, asterisks) or do not overexpress EGFP-Rab31. Scale bar = 20 μm. B, left panels, scrambled and GAPex5 siRNA-treated cells were immunostained for CD63 (pseudocolored green) along with EGF-TxR (red) and EGFP-Rab31 (pseudocolored white). The left bottom panels show individual and merged fluorescence signals of the boxed areas, magnified ×2. Box A encloses the central area of cells with Rab31 overexpression, whereas box B encloses central areas of cells with no Rab31 overexpression. Arrows indicate some structures positive for both EGF-TxR and CD63. Scale bar = 20 μm. Right panel, the percentage of EGF-TxR puncta that are also positive for CD63 were quantified and are graphically presented as a percentage of total EGF-TxR puncta counted. 41 cells in three independent experiments were analyzed, and data are shown as mean ± S.E. **, p < 0.01; Student's t test.