Fig. 3.

8ab protein is ER-luminal-oriented. (A). Diagram of constructs used for determining the orientation of 8ab within ER. A 3XFLAG epitope was tagged at the N-terminus of pCI8ab, and an HA epitope was tagged at the C-terminus. (B) 8ab is ER-luminal-oriented. HeLa cells were transiently transfected with pCI, pCI-3XFLAG8ab or pCI-8abHA as indicated. After 30 h, cells were fixed and subjected to total permeabilization with 0.05% saponin or selective permeabilization with 0.000125% digitonin, and then labeled with anti-Calreticulin, anti-HA and anti-FLAG antibodies. After mounting, cells were visualized by confocal microscopy.