Fig. 6

p53 interacts physically with murine α-syn promoter. a Scheme of the region of wild-type α-synuclein promoter (WT α-syn prom) with the p53 DNA-binding motif indicated in bold. The -970-967 (5’-CATG-3’) region underlined has been deleted (Δα-syn prom). b SH-SY5Y cells were co-transfected with either empty pcDNA3 vector (EV, lanes -) or p53 cDNA (p53, lanes +) and WT α-syn prom (black bars) or Δα-syn prom (empty bars) constructs then promoter activities were measured as described in the Methods section. p53 expression was controlled by anti-Flag antibodies as described in Methods. Bars represent the means ± SEM of 4 independent experiments performed in triplicates and are expressed as percentage of control EV-transfected cells. c EMSA analysis of the interaction of wild-type recombinant p53 (p53r) with α-syn-derived biotinylated probe encompassing the consensus sequence shown in (a). Reactions were carried out in absence (-) or in the presence (+) of either an excess of specific cold probes (comp-sp, see lanes 4, 5) or p53-directed antibodies (anti-p53 pab421/DO1, lanes 3, 6) and analyzed as described in the Methods. Free probe control is shown in lane 1. d ChIP analysis of the interaction between endogenous p53 with α-syn endogenous promoter in MEF cells by endpoint semi-quantitative PCR (upper gel) or real time PCR (histogram) as described in Methods. In (d), lanes 1–5 correspond to 100 bp DNA ladder (STD), normal mouse IgG ChIP (CT), p53 ChIP (IP), input (INP) and no template control (H₂O), respectively. Statistical analysis was performed with GraphPad Prism software by using either One-way ANOVA analysis of variance coupled to a Newman Keuls post-hoc test (b) or homoscedastic, unpaired Student’s t-test (d). Significant differences are: *p < 0.05, ***p < 0.001, and ns for non-significant