Supplementary Figure 8: Abnormal phosphorylation of GluR1 in Lrrk2–/– mice.
From: LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity
(a,b) Western Blot analysis of cultured LRRK2+/+ and LRRK2–/– cortical neurons treated with DMSO or FSK (50μM for 1hr) for the expression of phosphorylated (pS845) and total GluR1. The full-length images are shown in Supplementary Fig. 9a,b. (c) Western blot analyses show the effect of LRRK2 kinase inhibitor LRRK2-IN-1 on FSK-induced phosphorylation of GluR1 S845 and cofilin S3 in cultured cerebral cortical neurons. Culture cerebral cortical neurons (8DIV) derived from P0 Lrrk2+/+ mouse pups with or without 5μM LRRK2-IN-1 for 30min prior to DMSO or 50μM FSK treatment for additional 1h. The phosphorylation of LRRK2 S935 serves as a positive control for LRRK2-IN-1 inhibitory activity. The top band in the pS935 blot is a non-specific signal reactive to LRRK2 S935 antibody. The full-length images are shown in Supplementary Fig. 9c. (d,e) Bar graphs show alteration of GluR1 pS845 (d) and cofilin pS3 (e) in cultured cortical neurons treated with PKA activator FSK and LRRK2 inhibitor LRRK2-IN-1. Three independent cultures were used under each experimental condition. Data represent mean ± SEM. One-way ANOVA plus Tukey's Multiple Comparison Test, ****P<0.0001, ns: not significant. (f) Western blot analysis of pS831 and total GluR1 in the striatum of P21 Lrrk2+/+ and Lrrk2–/– mice after treated with saline (0) or Drd1 agonist SKF81297 at 2 and 10mg per kg bodyweight. The full-length images are shown in Supplementary Fig. 9d. (g) Quantification analysis of pS831 GluR1 ratio in the striatal tissues of P21 mice treated with SKF81297. N=4 per genotype. Data represent mean±SEM. Unpaired t-test, t(6)=0.2216, 1.413, and 1.223, respectively. (h-j) The full-length images of Fig. 6a,c, and f, respectively.
