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Fig. 3. hVPS4 function is required for the disassembly of bilayered clathrin coats on endosomal vacuoles. HeLa cells transfected with GFP, GFP-hVPS4^wt or GFP-hVPS4^EQ were double labeled for GFP (15 nm gold) and (A-C) Hrs (10 nm gold) or (D) clathrin (10 nm gold). (A) Cells expressing GFP (15 nm gold) show Hrs (10 nm gold) in the characteristic flat bilayered coats on endosomal vacuoles (EV). (B) Also, in hVPS4^wt cells, Hrs (10 nm gold) is present in flat bilayered coats. Inward budding profiles are formed next to the coat (arrow). (C,D) Cells overexpressing hVPS4^EQ show endosomal vacuoles positive for Hrs (10 nm gold in C) and clathrin (10 nm gold in D). The coat is assembled in an orderly fashion, displaying the characteristic two layers and label for hVPS4^EQ is present not within the coated areas but at the rims (arrowheads). Scale bars, 200 nm.

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