Figure 7.

Intracellular localization of internalized receptors in K562 cells transfected with α_Yβ_18-27 and incubated for 4 h at 37°C with or without 25 μM lactacystin or in cells expressing the ΔLys mutant. Cells were incubated for 30 min with 50 μM cycloheximide in the presence of 100 nM Tf-Cy5. After fixation and permeabilization, cells were first incubated with anti-chimera antibody (7G7B6) and with anti-Lamp-1 antibody (H4A3). The cells were then incubated with secondary labeled-antibody (anti-IgG2a-Texas Red and anti-IgG1-FITC) and analyzed by confocal microscopy. Merged images are shown where chimeric receptors are stained in green and the immunodetected intracellular markers (TfR or Lamp-1) in red. The yellow color indicates colocalization. (A) Control cells. (B) Cells expressing the ΔLys mutant. (C) Lactacystin-treated cells. Bar, 5 μM. (D) colocalization of the chimeric receptors with TfR or Lamp-1 was quantified as described in MATERIALS AND METHODS. (E) Colocalization of Lamp-1 with TfR in K562 α_Yβ_18-27 cells incubated with or without lactacystin_.-32767.