Supplementary Figure 4: PKA pathway is involved in LRRK2-dependent phosphorylation of cofilin.
From: LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity
(a) Western blot analysis of phosphorylated LIMK1/2 (p-LIMK1/2), total LIMK2, pSSH1L (S978), total SSH1L, PP1, and 14-3-3ζ proteins in the forebrain of P15 LRRK2+/+ and LRRK2–/– pups. The full-length images are shown in Supplementary Fig. 5a. (b) Quantification analysis of p-LIMK1 levels normalized to total LIMK1/2 levels derived of three independent experiments. Data represent min to max. Unpaired t-test, t(4)=1.087. (c) The level of phosphorylated SSH1L (p-SSH1L) normalized against the total SSH1L level in respective samples. Data represent min to max. Unpaired t-test, t(4)=1.087. N=3 per genotype and per time point. Data represent mean ± SEM. Unpaired t-test, t(4)=1.278. (d,e) Western blot analysis of cortical extracts derived of Lrrk2+/+ (d) and Lrrk2–/– (e) pups treated with indicated pharmacological compounds. The full-length images are shown in Supplementary Fig. 5b,c. (f, g) Bar graphs show quantification analysis of pS3/total cofilin levels. Bars represent mean± SEM (n=3). Unpaired t-test, *P<0.05, **P<0.01, ***P<0.001. (h) Western blot analysis of phosphorylated (p) and total CREB in cultured Lrrk2+/+ and Lrrk2–/– cortical neurons at 15DIV treated with DMSO or FSK. The full-length images are shown in Supplementary Fig. 5d. (i, j) Whiskers plots represent the quantification of pCREB levels normalized to total CREB levels (n=3 independent experiments per genotype). Data are represented as min to max. Unpaired t-test, Lrrk2+/+: t(4)=6.404, *p<0.05. Lrrk2–/–: t(4)=0.8727. (k) Western blot and quantification analysis of pCREB in the striatal nuclear extracts of Lrrk2+/+ and Lrrk2–/– mice at P15 (n=3 per genotype). The full-length images are shown in Supplementary Fig. 5e. (l) Whiskers plot shows min to max. Unpaired t-test, t(4)=4.310, *p<0.05. (m,n) The full-length images of Fig. 3a, 3c.
