Figure 7.

PLD2 acts downstream of the EFA6 mediated recycling of transferrin receptors. Cells were transfected with siRNA for PLD2 or a control siRNA, and 2 d later they were microinjected with a plasmid encoding GFP-EFA6. Cells were allowed to internalize fluorescent transferrin and were incubated in the absence of transferrin to allow internalized transferrin to recycle. Cells were fixed and stained with antibody to GFP to identify those overexpressing the exchange factor (A). Cells overexpressing EFA6 and treated with siPLD2 did not show the accelerated loss of fluorescent transferrin seen in cells treated with control siRNA. For quantification of transferrin, micrographs were taken using identical settings. Graph (B) shows average pixel intensity and SD of EFA6-expressing cells in control (n = 21) and siPLD2 (n = 15)-treated cells as well as that of neighboring uninjected cells (n = 34 from control samples and n = 14 from siPLD2 samples).