FIGURE 8.

PACS-2 and class I PI3K are required for Nef to down-regulate MHC-I in MEFs. A, Western blot (WB) of lysates from PACS-2^+/+ and PACS-2^-/- MEFs co-transfected (Amaxa) with pUHD10.1HLA-A2.1 and pNef-eYFP. B, after 48 h, anti-MHC-I (mAb W6/32, red) was added to the media for 1 h at 37 °C. Where indicated the cells were pretreated with 1 μm PI-103 or PIK-112 for 1 h prior to antibody uptake, and the inhibitors were maintained in the culture medium for the duration of the antibody uptake. Following uptake, the media were removed and replaced with fresh culture media for 30 min in the absence or presence of inhibitor. The cells were fixed, stained with a fluorescent secondary antibody, and then visualized by confocal microscopy. C, morphometric quantitation of internalized HLA-A2.1 (W6/32). The amount of internalized antibody was the same in WT or PACS-2^-/- cells transfected with pUHD10.1HLA-A2.1 alone, and therefore the same value was used for normalization of uptake in lane 1. The error bars represent the mean ± S.D. from 20 cells per marker from three independent experiments. Scale bar = 20 μm.