Fig 1.

AP-3 sorting machinery is impaired in HPS2 fibroblast cultures. (A) Western blot analysis of AP-3 expression in primary fibroblast cultures. The same number of normal and HPS2 fibroblast cultures was lysed. Four subunits of the AP-3 complex and actin were detected by immunoblotting using anti-δ, -β3A, -μ3A, -σ3A, and -actin antibodies. (B) Immunofluorescence microscopy analysis of the AP-3 complex in primary fibroblast cultures. Normal and HPS2 fibroblast cultures were transfected with a pECFP-Nuc plasmid. At 24 h after transfection, cells were fixed, permeabilized, and immunostained for the β3A subunit. pECFP-Nuc is shown in cyan, and the β3A subunit is in red. Bars, 10 μm. (C) Immunofluorescence microscopy analysis of subcellular localization of CD63 and LAMP-1. Normal (left) and HPS2 (right) fibroblast cultures were immunostained using anti-CD63 (top) and anti-LAMP-1 (bottom) antibodies. Bars, 10 μm. (D) Flow cytometric analysis of cell surface CD63 (top) and LAMP-1 (bottom). Unpermeabilized normal and HPS2 fibroblasts were immunostained with mouse anti-LAMP-3 or anti-LAMP-1 antibodies, followed by goat anti-mouse IgG conjugated to APC. The outlined plot represents the isotype control, the filled plot represents HPS2 fibroblast cultures, and the dash-lined plot represents normal fibroblast cultures.