Nef-eYFP and endogenous CI-MPR are mislocalized in
PACS-2-/- MEFs. A,
PACS-2+/+ and PACS-2-/- MEFs were transfected (Amaxa)
with pNef-eYFP. After 18 h the cells were fixed and stained with
anti-mannosidase II or anti-EEA1 (red) and visualized by confocal
microscopy. Western blot (WB) in upper right-hand corner of
PACS-2 in lysates from PACS-2+/+ and PACS-2-/- MEFs is
shown. Inset, Nef-eYFP in dispersed EEA1-postive compartments in
PACS-2-/- MEFs. B, PACS-2+/+ and
PACS-2-/- MEFs were transfected (Amaxa) or not with pGalT-CFP.
After 18 h the cells were fixed, stained with anti-EEA1 (red) or
anti-CI-MPR 8738 (red in GalT-expressing cells; green in
EEA1 co-stained cells (bottom)), and visualized by confocal
microscopy. Inset, CI-MPR in EEA1-positive compartments. Morphometric
analysis was performed as described under “Experimental
Procedures.” Error bars represent mean ± S.D. from 20
cells per marker in four independent experiments for Nef-eYFP and three
independent experiments for CI-MPR. Scale bar = 20 μm.