Sc4246

EMSA was performed by means of a commercial Gel shift Chemiluminescent EMSA Assay Kit provided by Active Motif. In brief, purified wild-type p53 recombinant protein (200 ng; SC4246, Santa Cruz) was incubated in 1X binding buffer (Promega) containing 100 ng poly (dI-dC), dithiothreitol (10 mM) and Bovine Serum Albumin (1 mg/ml) at 4 °C for 30 min with 20 fmol of double stranded 5’ biotin end-labeled SNCA-derived oligonucleotides (forward: 5′-CCT-TTC-GCT-GGA-GAC-ATG-CCC-TTC-CAT-CCT-GTC-AAA-GCC-C-3′; reverse: 5′-GGG-CTT-TGA-CAG-GAT-GGA-AGG-GCA-TGT-CTC-CAG-CGA-AAG-G-3′) encompassing the putative p53 responsive element. Protein-probe complexes were then resolved by electrophoresis on a 5 % native polyacrylamide gel at 4 °C, transferred to a positively charged nylon membrane (Thermo Scientific), cross-linked (UV-light cross-linker equipped with a 254 nm bulb) and revealed by means of streptavidin conjugated to horseradish peroxidase (HRP) and a chemiluminescent substrate. When indicated, in order to confirm specific DNA-protein interaction, we performed a 15–30 min pre-incubation at 4 °C with either an excess (4 pmol) of unlabeled specific and non-related control DNA or pab421 before adding the biotin-labeled probes.