Figure 5.

Trafficking of N528S-VWFpp and coexpressed mature VWF in AtT-20 cells. (A) AtT-20 cells were transiently transfected with wild-type VWFpp or N528S-VWFpp. Transfected cells were fixed and dual-stained for VWFpp (i, iv) and ACTH (ii, v) and examined by confocal microscopy. The merged image of the 2 stains is shown in the last column (iii, vi) with colocalization shown in yellow. Both wild-type VWFpp and N528S-VWFpp appear to traffic to endogenous ACTH-containing storage granules. (B) Wild-type VWFpp and N528S-VWFpp were coexpressed with propeptide-deleted VWF (Δpro) in AtT-20 cells. Transfected cells were dual-stained for VWFpp (vii, x) and VWF (viii, xi). The merged image shown in the last column (ix, xii) with colocalization shown in yellow. For wild-type VWFpp coexpressed with Δpro, both VWFpp and VWF were colocalized in granules, whereas for N528S-VWFpp with Δpro, only VWFpp was localized in granules, and VWF demonstrated diffuse staining. (C) Wild-type and N528S-VWF were expressed and cells were dual-stained for VWFpp (xiii, xvi, xix) and VWF (xiv, xvii, xx). The merged image shown in the last column (xv, xviii, xxi) with colocalization shown in yellow. For wild-type VWF, both VWFpp and VWF were colocalized in granules. Cells expressing N528S-VWF showed either granular VWFpp staining with diffuse VWF staining or diffuse staining for both proteins.