FIGURE 6.

PACS-1 and PACS-2 mediate the sorting of endocytosed CD8-CIMPR. HeLa-CD8CIMPR cells were nucleofected (Amaxa) with a control siRNA (scr) or siRNAs specific for PACS-1 or PACS-2. After 48 h, anti-CD8 (mAb UCHT-4, green) was added to the medium for 1 h at 37 °C. The media were removed and replaced with fresh culture medium for 30 min, and the cells were fixed and stained with anti-TfR (red) followed by isoform-specific fluorescent secondary antibodies. The cells were then visualized by confocal microscopy. Morphometric analysis was performed as described under “Experimental Procedures.” Error bars represent mean ± S.D. from 20 cells per marker in eight independent experiments. Bottom, Western blot (WB) of lysates from parallel samples of nucleofected cells. Scale bar = 20 μm.