Fig. 3

XBP1s SUMOylation increases in LRRK2-GS astrocytes. A Immunoprecipitation pulling down XBP1 in LRRK2-WT and -GS astrocytes co-transfected with different Myc-XBP1a mutant constructs and siRNA targeting 3ʹ-UTR region of XBP1s. SUMOylated XBP1 band intensity were quantified (lower graph). Data are means ± SD (*p < 0.05, **p < 0.01). B Astrocytes from LRRK2-WT and -GS were treated with tunicamycin and then subjected to immunoprecipitation with an antibody against XBP1 or PIAS1. SUMOylated XBP1 and XBP1-PIAS1 interacting band intensities were quantified (lower graph). Data are means ± SD (*p < 0.05). C, D LRRK2-WT and -GS astrocytes were transfected with PIAS1-specific siRNA (si-PIAS1) and then stimulated with tunicamycin. Protein interactions were assessed by immunoprecipitation with an antibody against XBP1 (C), and XBP1–SUMO1 interactions were detected using the PLA method (D). SUMOylated XBP1 and XBP1-PIAS1 interacting band intensity were quantified (lower graph). Data are means ± SD (*p < 0.05, **p < 0.01). PLA signals were quantified and displayed graphically as the average number of spots. Ten fields of view per group of three independent experiments (n = 30). Scale bar, 20 μm. Data are means ± SD (**p < 0.01). Open arrowhead denotes SUMOylated XBP1 and black arrowhead indicates non-modified XBP1. All band intensities were quantified using an Image J program