Figure 1

Expression of the proline rich region of POB1 has a dominant negative effect on EGF endocytosis but it does not affect transferrin endocytosis. HeLa cells were transiently transfected with GFP-POB1 PRD1 (308–365) or GFP-POB1 full length or, as controls, GFP-Dynamin K44A or GFP alone. Cells were serum starved and incubated with TRITC labeled EGF for 20 minutes at 37°C. Cells were fixed, without acid wash, and observed on an Olympus IX 70 with Nanomover^® and softWoRx Delta Vision. Transfected cells are visible in the 528 nm channel as green colored. TRITC-EGF was visualized at 617 nm and is colored in red. Endocytosed vescicles with TRITC-EGF, visible as fluorescent spots, are not detectable in cells over expressing the mutated Dynamin K44A or the central proline rich region of POB1 (POB1 PRD1). A black arrow points the transfected cells in the zoomed sections. HeLa cells transfected with GFP-POB1 PRD1 (308–365) were serum starved and incubated with TRITC-Transferrin (100 mg/ml). POB1-PRD1 transfected cells are visualized as above, internalized transferrin is visible as fluorescent red spots.