Fig 1.
(A) Western blot analysis of PGRN levels in BV2 cells expressing control or guide RNA to the mouse GRN gene. (B) Schematic illustration of the SILAC experiment searching for PGRN interactors. (C) Volcano plot of SILAC hits. Top hits identified in the heavy fraction are highlighted. (D) Anti-PGRN co-immunoprecipitation of FLAG-PGRN and GBA-myc overexpressed in HEK293T cells. (E) Anti-PGRN co-immunoprecipitation of PGRN and GBA-myc overexpressed in HEK293T cells using buffers of pH7.5 and pH5.3.
Fig 2.
Interaction between GCase and granulin peptides.
(A) Anti-GFP co-immunoprecipitation of GFP-PGRN or individual GFP-granulins and GBA-myc overexpressed in HEK293T cells shows binding primarily to Grn F, with weaker binding to Grn A and other Grn peptides. (B) Anti-GFP co-immunoprecipitation of GFP-PGRN or individual GFP-granulins and GBA-myc from conditioned medium of transfected cells shows weaker binding to all the granulins except Grn G.
Fig 3.
PGRN deficiency results in decreased GCase activity in mouse tissues.
GCase activity was assessed in tissue lysates from 2-month-old WT and Grn-/- mice, as indicated, using the substrate 4-MU. (n = 5–6, ±SEM, *p value <0.05, **p-value <0.01, ns, not significant, Student’s t-test.
Fig 4.
PGRN deficiency results in decreased GCase activity without changes in GCase protein levels.
Tissue lysates from 2-month-old WT and Grn-/- mice were incubated with the MDW941 GCase activity probe. The samples were run on SDS-PAGE and MDW941 labeled GCase (active) was detected using a fluorescent scanner, then western blot was performed to assess total GCase protein levels. n = 5–6, ±SEM, *p value <0.05, **p-value <0.01, ns, not significant, Student’s t-test.
Fig 5.
GCase is enriched in the lysosome fraction of WT and Grn-/- livers.
Lysosomes were isolated from liver tissues of 2-month-old WT and Grn-/- mice. The presence of GCase, LAMP1 and calnexin (marker for ER) was determined in the different fractions using Western blot analysis.
Fig 6.
PSAP and total saposin levels are not changed in PGRN deficient mice.
Mouse tissue lysates from 2-month-old WT and Grn-/- mice were assessed for PSAP and total saposin peptide levels, which were normalized to GAPDH. n = 5–6, ±SEM, ns, not significant, Student’s t-test.