Supplementary Figure 6: LRRK2 regulation lies downstream of cAMP production.
From: LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity
(a) cAMP levels in cultured Lrrk2+/+ and Lrrk2–/– cortical neuronal extracts (n=3 independent experiments per genotype with duplication of each sample). Data are represented as mean ± SEM. Unpaired t-test, t(4)=1.771, p=0.1020. (b) Cultured cortical neurons treated with vehicle (DMSO) or FSK were subject to cAMP production ELISA assay. Bar shows the response to FSK as a percentage changes relative to the average value of DMSO treated neurons from three independent experiments. (c) Western blot illustrating PSD95, LRRK2 and PKARIIβ protein levels in different subcellular fractions (described in detail in Materials and Methods section) of both Lrrk2+/+ and Lrrk2–/– brains. The full-length images are shown in Supplementary Fig. 7a. (d) Striatal extracts at different ages during development were tested for the presence of pPKARIIβ, PKARIIβ, and PKACα in both genotypes. The full-length images are shown in Supplementary Fig. 7b. (e) Co-IP of MBP-tagged PKARIIβ, GST-tagged LRRK2 and PKACα recombinant proteins using a PKACα antibody. LRRK2 does not affect the interaction between PKACα and PKARIIβ. The full-length images are shown in Supplementary Fig. 7c. Four independent experiments were performed. Unpaired t-test, t(6)=1.5685, p=0.1329. (f) Co-staining of endogenous PKACα (green) and PKARIIβ (red) in cultured Lrrk2+/+ and Lrrk2–/– hippocampal neurons at 15DIV. Scale bar: 10μm. (g-m) The full-length images of Fig. 4a-e, 4h, and 4i.
