Figure 3.

PSAP is required for PGRN lysosomal targeting in fibroblasts. (A) Mislocalization of PGRN in PSAP^−/− fibroblasts. Immunostaining for PGRN, LAMP1, and PSAP in fibroblasts derived from wild-type and PSAP^−/− mice using sheep anti– mouse PGRN, rat anti–mouse LAMP1, and rabbit anti–mouse PSAP antibodies. PGRN mislocalization was observed in all the PSAP^−/− fibroblasts examined. (B) Quantification of PGRN localization within LAMP1-positive vesicles in wild-type and PSAP^−/− fibroblasts using ImageJ. Data are presented as mean ± SEM from three independent experiments. ***, P < 0.001, Student’s t test. (C) Increased PGRN secretion in PSAP^−/− fibroblasts. Fibroblasts were cultured in serum-free medium for 48 h before the lysates and conditioned media (CM) were harvested. Proteins from the conditioned media were precipitated with TCA. (D) Quantification of experiments in C. PGRN levels are normalized to transferrin in the conditioned media and Gapdh in the cell lysates. Data are presented as mean ± SEM from four independent experiments. *, P < 0.05, Student’s t test; N.S., no significance. (E) Immunostaining for PGRN, PDI, and cathepsin D in fibroblasts derived from wild-type and PSAP^−/− mice. Representative images from three replicated experiments are shown Bars: (main) 20 µm; (inset) 5 μm.