Figure 3

HuPAR topology. A. HuPAR-2 topology model derived by hydrophobicity algorithms and the experiments described in panel B-D is depicted. B-C. HuPAR-2 bearing an N- or C-terminal HA-tag was transiently transfected into 293T cells. After 48 hours cells were treated with saponin (intracellular staining) or without (surface staining). Following immunostaining using an anti-HA antibody and a FITC-conjugated secondary antibody, the samples were visualised either by confocal microscopy (B) or processed by flow cytometry (C). Immunostaining of the cells with anti-human CD71 was used as cell surface protein control. The cells nuclei were counter stained with propidium iodide. D. Cell lysates from 293T transiently transfected with an empty pcDNA3 (-), HuPAR-2 (wild type), HA-tagged HuPAR-2 wild type (C-HA wild type) or glycosylation mutant (C-HA N178A) were either treated (+) or untreated (-) with an enzyme removing N-linked oligosaccharide chains (PNGase F) and analysed by western blotting.