Fig. 6: GCase activity and level is reduced in lysosomal extracts from LRRK2 KO RAW264.7 cells.

a RAW264.7 cells, WT and LRRK2-deficient (KO) were processed for western immunoblotting of the total cell extract and crude lysosomal fraction, and the membranes probed for LRRK2, GCase, and γ-tubulin. GCase levels are unchanged in the cell lysates of LRRK2 KO cells, compared to WT cells; however, they are reduced (though non-significantly) in crude lysosomal fractions from KO RAW264.7 cells. On the right, quantification of GCase levels (n = 6 independent replicates, mean ± SEM: WT = 0.03441 ± 0.01905, KO = 0.01460 ± 0.007323; p = 0.0625, Wilcoxon matched-pairs signed rank test, effectiveness of pairing p = 0.0417) and activity normalized by GCase levels (WT = 28121 ± 8744, KO = 17777 ± 5171; p = 0.0625, Wilcoxon matched-pairs signed rank test, effectiveness of pairing p = 0.0083; mean ± SEM) in the lysosomal fraction. b Western blot analysis of LRRK2 WT vs. KO RAW total lysates of pS935 LRRK2, total LRRK2 (MJFF3 antibody), GCase and β-actin loading control. Treatment with 100 nM MLi-2 for 90’ results in complete dephosphorylation of S935 as expected (n = 3 biological replicates each with 2 technical replicates; two-way ANOVA, Tukey’s multiple comparisons test, WT DMSO vs. WT MLi-2 p = 0.3799; KO DMSO vs. KO MLi-2 p = 0.0995; genotype effect p = 0.4325 F(1, 20) = 0.6417, treatment effect p = 0.5732 F(1, 20) = 0.3280). c Quantification of GCase levels does not show differences across genotypes and treatments (two-way ANOVA, Tukey’s multiple comparison’s test; p > 0.05; data are expressed as mean ± SEM). d GCase activity assessed with the in-lysate 4-MU protocol comparing WT vs. KO RAW cell lysates treated for 90’ with DMSO, 10 or 100 mM MLi-2 (n = 2 biological replicates each with 2 technical replicates, two-way ANOVA, Tukey’s multiple comparison’s test; WT:DMSO vs. KO:DMSO p = 0.0388; WT:DMSO vs. KO:10 nM MLi-2 p = 0.0191; WT:DMSO vs. KO:100 nM MLi-2 p = 0.0413; WT:DMSO vs. WT:100 nM MLi-2 p = 0.25; genotype effect p = 0.0051 F(1, 18) = 10.15, treatment effect p = 0.1416 F(2, 18) = 2.184; data are expressed as mean ± SEM). e GCase activity assessed with the in-cell protocol from Benz et al. (2021) comparing WT vs. KO RAW cell lysates treated for 90’ with DMSO, 100 nM or the GCase inhibitor CBE (n = 3 biological replicates with 3–4 technical replicates, two-way ANOVA, Tukey’s multiple comparison’s test; WT:DMSO vs. WT: 100 nM MLi-2 p = 0.0004; KO:DMSO vs. KO:100 nM MLi-2 p = 0.3883; WT:DMSO vs. KO:DMSO p = 0.3901; other non-informative comparisons, both p > 0.05 and <0.05, were omitted; genotype effect p = 0.7918 F(1, 54) = 0.07040; treatment effect p < 0.0001 F(2, 54) = 8.28; data are expressed as mean ± SEM).