Fig. 4. Characterization of the Gas3/PMP22-positive vacuoles. (A) NIH3T3 or IMR90-E1A cells were microinjected with Gas3/PMP22-GFP and 12 hours later were fixed and probed with antibodies to annexin II, Tfn-R, and P0 or stained with WGA as indicated. Endocytosis of biotinylated Tfn was performed as described in Materials and Methods. The arrowhead indicates vacuoles negative for annexin II staining, whereas arrows point to vacuoles negative for Tfn-R and Tfn staining. Bar, 8 µm. (B) Dynamin1 wt and the point mutated derivative K44A defective in GTP binding and hydrolysis were co-expressed with Gas3/PMP22-GFP. After 18 hours from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22-GFP and dynamin as described in Materials and Methods. Cells were scored for accumulation of vacuoles. Data represent arithmetic means±s.d. for four independent experiments.