Fig 2: Analysis of AnxA2 function in G2019S primary striatal astrocytes at endogenous level. A Western blot analysis of primary striatal astrocyte lysates under PFF-treated and basal conditions using anti-Lrrk2, anti-a-syn, and anti-AnxA2 antibodies. B, C, D Quantification of band intensity was performed using ImageJ and normalized by ß-actin (n = 4). E Representative images of Lrrk2+/+ and Lrrk2GS/GS astrocytes treated or not with a-syn PFFs and stained with anti-AnxA2 (green), anti-a-syn (red), F-actin (cyano), and cell nuclei with DAPI (blue). MLi-2-treated Lrrk2GS/GS astrocytes are shown in the bottom panels. Scale bar 20 µm. Insets show a close-up of a-syn inclusions and re-localized AnxA2. F, G Eight images per cell culture were analyzed (n = 3). Quantifications of Anxa2 puncta and AnxA2-a-syn PFFs proximity were performed using ImageJ. Statistical analysis in F and G was made by Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Statistical analysis in B, C, and D was performed with unpaired t test or one-way ANOVA followed by Tukey’s multiple comparisons test. *p = 0.05, **p = 0.01, ***p = 0.001

Fig 3: Characterization of LRRK2 interactoma in stimulated condition. A Schematic outline of the experimental setup. H4 cells were transfected using 3xFlag-LRRK2 encoding plasmid and, after 48 h post transfection, treated with 0.5 µM sonicated unlabeled a-syn PFFs for 24 h. LRRK2 was subsequently immunopurified using anti-Flag agarose beads (IP), eluted with Flag peptide (elution), and subjected to LC-MS/MS analysis. FT, flow-through; unbound LRRK2. B Western blot analysis showing LRRK2 expression, immunopurification, and elution in H4 cells in treated and basal conditions. C Relative quantification of LRRK2 interactome under treated and untreated conditions. The area of the precursor ions identified by LC-MS/MS analysis was used as a quantitative measure of the protein content. The ratio between the area of the precursor ions of untreated and treated samples (normalized by the content of LRRK2) was then considered to highlight proteins showing a different affinity for LRRK2 in the two conditions (n = 2). D H4 cells transfected with GFP-ANXA2 in a-syn PFF-treated condition verifying the proximity of internalized a-syn fibrils and transfected AnxA2. AnxA2-GFP (green), a-syn (red), DAPI (blue). Scale bar 30 µm