Fig. 3. The effects of wortmannin on intracellular trafficking mimic those of Rab5 Q79L. (A) Regulation of the EGF-induced lysosomal degradation of EGFR by Rab5. 293T cells were transfected with plasmids expressing wild-type Rab5, mutant Rab5 S34N or Q79L for 48 h and then stimulated with EGF for the indicated times. Cell lysates were subjected to immunoblotting analysis with anti-EGFR or anti-Rab5 antibodies. (B and C) The effects of wortmannin and Rab5 on endosome morphology. BT20 (B) and SKBR-3 (C) cells were transfected with wild-type Rab5 or mutant Rab5 Q79L for 48 h. Cells were then treated with wortmannin and stimulated with EGF for 30 min or not stimulated. EGFR and Rab5 were detected by indirect immunofluorescence. Photographs are double exposures with EGFR localization in the red channel (arrows) and Rab5 localization in the green channel (arrowheads) for BT20 cells, and with EGFR localization in the green channel (arrows) and Rab5 localization in the red channel (arrowheads) for SKBR-3 cells. Inset: co-localization of EGFR and Rab5 in endosomes. Size bar = 20 µm.