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Fig. 3 | Molecular Neurodegeneration

Fig. 3

From: A role for α-Synuclein in axon growth and its implications in corticostriatal glutamatergic plasticity in Parkinson’s disease

Fig. 3

α-Syn expression regulates PI4,5P2 levels. a SK-mel2 cells transduced with viral particles expressing either sh-SNCA or a control (sh-Cntrl). 3–7 days post transduction, samples of lysed cells (80 μg protein) were analyzed by Western blotting for α-Syn expression using MJFR1 antibody. b SK-mel2 cells transduced as in (a) and analyzed by FACS for α-Syn, using anti α-Syn antibody MJFR1; and c using anti PI4,5P2 antibody. Mean ± SE, n = 4000 cells; *, P < 0.05, ttest. d HEK 293 T cells were transfected either with WT α-Syn, A53T or K10,12E α-Syn mutations, or mock transfected. 48 h post transfection, PI4,5P2 levels were determined by FACS, using anti PI4,5P2 antibody. Cells were gated based on positive signal for α-Syn detected by MJFR1 antibody. Mean ± SE, n > 3000 cells; *, P < 0.05, one way ANOVA. e HeLa cells, co-transfected with WT α-Syn and PH-PLCδ1-GFP. Control cells transfected with the mock plasmid together with PH-PLCδ1-GFP. Cells were incubated with Alexa-647 Concanavalin (Con) A for visualization of plasma membrane (PM) and processed for ICC with anti α-Syn antibody, C20. Showing the co-localization of PH-PLCδ1-GFP and 647-ConA signals (direct fluorescence). Bar = 10 μm. f Calculated plasma membrane to cytosol ratio of the PH-PLCδ1-GFP signal in HeLa cells, co-expressing the indicated constructs. Mean ± SE of n = 15-25 cells. *, p < 0.05, one way ANOVA. g High magnification of paraffin embedded mouse brain section containing the hippocampus, analyzed by IHC and immunoreacted with anti PI4,5P2 ab (green). Section stained with DAPI (blue). Nomarski image (gray) shown to demonstrate PI4,5P2 signal on PM. Bar = 10 μM. h Primary cortical neurons from WT C57BL/6 or α-Syn −/− (C57BL/6JOlaHsd) at 14 DIV. Cultured neurons analyzed by ICC and immunoreacted with anti NF-200 and anti PI4,5P2 abs. Bar graph shows signal ratio of PI4,5P2 to NF200. Mean ± SD, n = 5 fields, consisting of > 10 neuronal cell bodies each; *, P < 0.05, t-test

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