FIGURE 6:

COPI binding motif alters the intracellular distribution of ERManI. (A) Wild type or the ERManI^COPI+ was transfected into NIH/3T3 cells. After culture on coverslips for 48 h, the cells were fixed and coimmunostained with anti-ERManI mAbs and anti-PDI or anti-β-COP antibodies. ERManI was visualized using Alexa 488–conjugated anti–mouse secondary antibody (green). PDI or β-COP was visualized using Alexa 555–conjugated anti–rabbit secondary antibodies (red). Cell nuclei were counterstained with To-PRO3. The inset on the bottom of each merged image shows a highly magnified view of the squared area. Arrowheads point to the cells transfected with wild type or mutant ERManI. Arrows show the colocalization of ERManI^COPI+ with PDI or β-COP. (B) NIH/3T3 cells transfected and stained as in (A) were counted double-blinded in three repeated experiments. The bar graph shows the percentage of cells showing ERManI localization solely in the Golgi (Golgi) or in both Golgi and ER (Golgi+ER). Error bars represent SD from three repeated experiments.