Figure 5.

PSAP facilitates PGRN lysosomal targeting from the extracellular space. (A) PSAP facilitates PGRN lysosomal targeting from the extracellular space in fibroblasts. GRN^−/− fibroblasts were treated with recombinant human his-PSAP and human FLAG-PGRN-his at a concentration of 5 µg/ml in serum-free media for 12 h as indicated. Fixed cells were stained with goat anti–human PGRN, rat anti–mouse LAMP1, and rabbit anti–human saposin B (PSAP) antibodies. Representative images from three replicated experiments are shown. Bars: (main) 20 µm; (inset) 5 μm. (B) Western blot analysis of PGRN and PSAP proteins in the uptake assay. GRN^−/− fibroblasts were treated with PGRN and PSAP proteins as in A and the proteins in the lysate after 24-h uptake as well proteins in the medium before and after 24-h uptake are shown. Western blots were detected using goat anti–human PGRN and rabbit anti–human saposin B antibodies. For unknown reasons, PSAP signal is always stronger in the medium when PGRN is added together in the Western blot (although the same amount of PSAP protein was added). (C) Quantification of PGRN–PSAP levels in the medium for experiment in B. Data are presented as mean ± SEM from three independent experiments. ***, P < 0.01, Student’s t test; N.S., no significance.