Fig. 3. LC3 is found associated with autophagosome membranes in addition to the cytoplasm. (A–E) HeLa cells transfected with GFP–LC3 were incubated at 37°C for 8 h in the presence of 10 mg/ml HRP and were cultured in Hanks’ solution at 37°C for 60 min. The cells were fixed and stained with DAB to detect the endocytosed HRP. Then, the localization of GFP–LC3 in the cells was examined by silver-enhanced immunogold electron microscopy using antibody against GFP. Arrowheads and arrows in (A–E), respectively, indicate autophagosomes and autolysosomes containing HRP. Bar, 1 µm. (F and G) ES cells were cultured in Hanks’ solution at 37°C for 1 h and fixed. The localization of endogenous LC3 was examined by silver-enhanced immunogold electron microscopy using antibody against LC3. The open and closed arrowheads indicate LC3 associated with inner and outer membranes of the autophagosome, respectively. Bar, 1 µm.