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. 2019 Oct 10;29(5):716–726. doi: 10.1093/hmg/ddz229

Figure 3.

Figure 3

GCase activity is reduced in iPSC-derived PGRN mutant neurons and can be rescued by increasing saposin C. (A) Western blot of total lysate input and lysosome-enriched samples of PGRN WT and mutant neurons (day 65 post-differentiation) (n = 3). (B) Quantification of GCase activity assay using lysates of lysosome-enriched samples from PGRN WT and mutant neurons (n = 3). GCase activity assay using recombinant GCase incubated with recombinant PGRN at (C) pH 5.9 or (D) pH 4.8 with 0.01% phosphatidylserine (n = 3). (E) Western blot analysis of PGRN WT and mutant neuron lysates treated with Endo H, PNGase F or untreated control. (F) Confocal images of PGRN mutant neurons showing colocalization of Alexafluor-488 labeled saposin C with lysotracker positive compartments after 3 hour treatment with saposin C (200 ng/ml) (n = 3). (G) Quantification of lysosomal GCase activity in PGRN WT and mutant neurons with and without treatment with recombinant saposin C (200 ng/ml). Scale bar = 10 μm. Data are presented as mean ± SEM, n.s. (not significant), *P < 0.05, **P < 0.05, (A) two tailed Student’s t-test, (G) one-way ANOVA followed by Tukeys multiple comparisons post hoc test.