Figure 4

GGA overexpression perturbs cellular sorting pathways. (A) HeLa cells were transfected with Myc-tagged GGA expression vectors. 24 h posttransfection, cells were washed and incubated for 30 min at 4°C in SFM containing 10 μg/ml Tf-594 then incubated at 37°C for 15 min. Cells were fixed and stained with anti-Myc antibody for transferrin binding and uptake assay. Lower panel: control untransfected cells were incubated with Tf-594 as indicated above and fixed immediately (0 min) or after a 15 min chase. (B) HeLa cells transfected with GGA expression constructs were stained with anti-TfR antibody followed by fluorescence microscopy. Lower panel, untransfected cell control. (C) HeLa cells were transfected with Myc-tagged GGA expression constructs, stained with anti-CD-MPR antibody followed by microscopy. Numbers represent average colocalization (R value) ± SD from 10 cells. Size bar represents 15 μm.