FIGURE 2.

Hhat topology mapping using FLAG insert constructs. COS-7 cells were transfected with Hhat cDNA containing the indicated internal FLAG epitope tags. Digitonin was used to selectively permeabilize the plasma membrane, and Triton X-100 was used to permeabilize all cellular membranes. Cells were stained with anti-FLAG antibody to visualize the cytosolic or lumenal orientation of the inserted FLAG tag. Staining of endogenous PDI served as an internal control for a lumenal epitope. Images for each construct were collected using the same conditions on the same day ensuring fair side-by-side comparison.