Fig. 2. DN-HA-14-3-3 proteins form inactive dimers and cannot interact with PI4KIIIß. (A) Left panel: HEK293 cells were cotransfected with plasmids encoding wildtype YN-14-3-3/YC-14-3-3 (red) or YN-14-3-3/YC-DN-14-3-3 (blue). Cells were harvested 48 hours after transfection in PBS supplemented with 5% FCS and 0.05% sodium azide, and BiFC was analysed by fluorescence flow cytometry. Untransfected cells were used as a negative control (filled). The data are presented in a histogram depicting EYFP fluorescence (FL1) (x axis) vs. cell number (count) (y axis). The results are representative of three independent experiments. Right panel: COS7 cells were cotransfected with the indicated plasmids. Cells were fixed 2 days after transfection and BiFC was analysed with confocal microscopy. A single optical section is shown. (B) Flag-tagged wildtype PI4KIIIß expression vectors were transiently transfected into HEK293 cells. Cells were lysed and Flag-PI4KIIIß proteins were precipitated with wildtype GST-14-3-3 or DN-GST-14-3-3 coupled Glutathione sepharose beads, resolved by SDS-PAGE and detected with Flag-specific antibodies. Equal amounts of GST-fusion proteins were visualized with anti-GST antibodies. (C) Left panel: Flag-tagged wildtype PI4KIIIß expression vector was transiently transfected into HEK293 cells along with HA-tagged 14-3-3 or HA-tagged DN-14-3-3, respectively. PI4KIIIß was precipitated using Flag-specific antibodies and protein complexes immunoblotted with a HA-specific monoclonal antibody. To verify HA-14-3-3 expression, total cell lysates were immunoblotted with Flag- and HA-specific antibodies. Right panel: HEK293 cells were transfected with HA-tagged wildtype or DN-14-3-3 expression vectors. 14-3-3 was precipitated using HA-specific antibodies and protein complexes immunoblotted with a PI4KIIIß-specific monoclonal and a 14-3-3-specific polyclonal antibody. Bar, 10 µm. PD, pulldown.