Figure 2. PGRN facilitates uptake and lysosomal targeting of PSAP in primary cortical neurons.

(a) Primary cortical neurons (DIV12) were incubated with AP-PSAP (50 nM) alone, or together with purified recombinant his-PSAP (10 μg ml⁻¹), or his-PGRN (1 μg ml⁻¹) as indicated. Scale bar, 50 μm. (b) Quantification of bound AP intensity of (a); n=3, ***P<0.001, **P<0.01, one-way analysis of variance (ANOVA). Data are presented as mean ±SEM. (c) Primary cortical neurons (DIV12) were treated with recombinant hPSAP (1 μg ml⁻¹) and/or hPGRN (1 μg ml⁻¹) for 16 h as indicated. Cells were stained with anti-mouse LAMP1, anti-human saposin B and anti-human PGRN antibodies. Scale bar, 20 μm. Representative images from three independent experiments were shown. (d) Primary cortical neurons (DIV12) were treated as in c. The cells were collected and subjected to immunoblotting with anti-human saposin B, anti-human PGRN and anti-β III tubulin antibodies. (e) Quantification of endocytosed neuronal PSAP and PGRN in (d), normalized to PSAP or PGRN alone. n=3, **, p<0.001, *, p<0.05, paired t-test. Data presented as mean±SEM. ***P<0.001, Student's t-test.