Fig. 4
α-Syn effect to elongate axons and collaterals is mediated through PI4,5P2. a PI4,5P2 levels determined by FACS, in HEK 293 T cells, following 48 h from cell transfection with α-Syn and/or Sj-1 phosphatase, as indicated. Mean ± SD of two independent experiments, n > 3000 cells in each treatment; P < 0.05, one-way ANOVA. b PI4,5P2 levels determined by FACS in HEK 293 T cells transfected with α-Syn and/or PIPKIγ as indicated. Mean ± SD of two independent experiments, n > 3000 cells in each treatment; P < 0.05, one-way ANOVA. c Primary cortical cultures from α-Syn−/− mouse brains, electroporated at day of preparation to co-express WT α-Syn together either with PIPKIγ-GFP, Sj-1-GFP or mock-GFP expressing vectors (as indicated). Cultures were fixed and immunoreacted with anti α-Syn MJFR1 ab and α-tubulin. GFP signal was captured by direct fluorescence. α-tubulin signal is shown. Bar = 25 μm. d The average length of axons (in μm). e Total length of collaterals per axon (in μm); and f PI4,5P2 levels within the axon and its collaterals (per axon area) quantified by Fiji program. Mean ± SE; n > 25 cells; *, P < 0.05 one-way ANOVA. g HEK 293 T cells transfected to express mCherry-Nir2 or mock transfected (cntrl). Western blot showing mCherry-Nir2 signal detected with anti Nir2 ab (Abcam). PI4,5P2 levels determined by FACS. Mean ± SE, n > 3000 cells; *, P < 0.05, ttest. h Tet-on inducible SH-SY5Y cells for inducible α-Syn expression were infected with viral vectors expressing either shNIR2 or shCntrl (Mission, Sigma-Aldrich). α-Syn expression induced with doxycycline (1 μM/ml) or non-induced. PI4,5P2 levels determined by FACS. Mean ± SD of n = 3 different experiments, > 3000 cells in each treatment. i Quantitative PCR (qPCR) detection of α-Syn following its induced expression in SH-SY5Y cells without and with the addition of doxycycline for 72 h. α-Syn mRNA levels normalized to the levels of G6PD gene detected in the same sample. j Quantitative detection of NIR2 expression by qPCR in these cells