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. 2013 Dec 31;289(7):4161–4172. doi: 10.1074/jbc.M113.529271

FIGURE 2.

FIGURE 2.

ApoER2 and VLDLR internalize clusterin via the early endosomal compartment. A, 3T3 cells expressing ApoER2 (ApoER2 3T3), (B) VLDLR (VLDLR 3T3), or (C) no receptor (3T3) were incubated with DyLightTM 488 labeled clusterin (green) at 4 °C to allow binding of clusterin to the receptors. ApoER2 3T3 (D–F), VLDLR 3T3 (G–I), or 3T3 (J–L) were incubated with DyLightTM 594-labeled clusterin (red) at 4 °C. After 1 h, the clusterin-containing medium was replaced with normal medium, and the cells were shifted to 37 °C to allow uptake of the ligand. Immunostaining with a goat anti-EEA1 antibody to visualize the early endosomal compartment is shown in green (E, H, K). Clusterin (red) colocalizes with the early endosome (green) in ApoER2 3T3 (F) and VLDLR 3T3 (I) but not in 3T3 cells (L). Experiments were repeated three times (A–C) or four times (D–L) with similar results. More than 30 cells were analyzed per condition. Scale bars represent 10 μm.