Fig. 2

Transcriptional activity of XBP1 is reduced in LRRK2-GS astrocytes. A LRRK2-WT and -GS astrocytes were treated with tunicamycin. Cell lysates were subjected to chemical crosslinking (XL) with disuccinimidyl suberate (DSS), to stabilize IRE1 oligomers, and were analyzed by WB. B XBP1 mRNA splicing was analyzed by RT-PCR using total RNA isolated from LRRK2-WT and -GS astrocytes. The ratio of XBP1 was quantified (right panel). C Western blot analysis of XBP1 in the cytosol and nuclear fractions of LRRK2-WT and -GS astrocytes. Gapdh and Sp1 were used as cytosolic and nuclear loading controls, respectively. D Luciferase reporter assay in LRRK2-WT and -GS astrocytes. Luciferase gene expression was driven by a synthetic promoter consisting of four tandem XBP1-binding sites. Data are means ± SD of three independent experiments (*p < 0.05). E Cell lysates from LRRK2-WT and -GS astrocytes were immunoprecipitated with anti-XBP1. The XBP1-bound DNA was recovered, and qRT-PCR were performed for indicated genes promoter region (upper). Expression levels of the indicated mRNAs was analyzed by real-time qPCR (lower). Data are presented as means ± SD of four independent experiments (*p < 0.05)