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. 2020 Nov 25;19(9):1546–1560. doi: 10.1074/mcp.RA120.002055

Fig. 4.

Fig. 4

Rab10-pThr73 occupancy in PD patient-derived neutrophils.A, Immunoblotting of neutrophils isolated from four PD patients with the G2019S LRRK2 mutation and four healthy controls (−/+100 nm MLi-2, 30 min) using anti total LRRK2, pSer935 LRRK2, total Rab10, MJFF-pRAB10 (pThr73) and GAPDH antibodies. B, Quantitation of immunoblots by analyzing phospho-Thr73 Rab10/total Rab10 ratio (right) and phospho-Thr73 Rab10/GAPDH ratio (left panel). C, Blind application of our mxSIM assay to the same mock (black) and MLi2-treated (grey) lysates (n = 3). Normalized Rab10- pThr73 occupancies by subtracting the MLi-2 treated values are also shown (red). Error bars represent SEM. D, Quantification of Rab10-pThr73 occupancy in controls and PD patients with the G2019S LRRK2 mutation. Each data point represents the median of triplicate measurements of untreated samples that are normalized to MLi2. One-way ANOVA with Bonferroni's multiple comparisons test was applied and the occupancies are presented as means ± SEM. E, Pearson correlation between immunoblotting and mxSIM assay. Plotted are fold changes of normalized Rab10-pThr73 levels (occupancies in mxSIM) relative to control cases. F, Immunoblotting of neutrophils isolated from controls, idiopathic PD patients and heterogeneous VPS35 D620N mutation carriers (−/+ 200 nm MLi-2, 30 min) using anti-total LRRK2, pSer935 LRRK2, total Rab10, MJFF-pRAB10 (pThr73) and GAPDH antibodies. G, Quantitation of immunoblots by analyzing phospho-Thr73 Rab10/total Rab10 ratio (right) and phospho-Thr73 Rab10/GAPDH ratio (left panel). H, Blind application of our assay to the same mock (black) and MLi2-treated (grey) lysates (n = 3). Normalized Rab10- pThr73 occupancies by subtracting the MLi-2 treated values are shown in red. Error bars represent SEM. I, Quantification of Rab10-pThr73 occupancy in controls, idiopathic PD and PD patients with heterogeneous VPS35 D620N mutation. The significant analysis was done as in (D). J, Same as (E) with healty controls (HC), idiopathic PD (iPD) and VPS35 D620N (VPS35). K, Quantification of Rab10-pThr73 occupancy between controls, idiopathic cases and PD patients with a defined mutation. The significant analysis was done as in (D).