Figure 3

Effect of NO on subcellular localization of parkin and its physical interaction with p53 promoter (Pp53). A, Histological images of SH-SY5Y cells transiently transfected with myc-tagged parkin and exposed to 200 μM old or fresh SNOC. Cells were probed with anti-myc antibody (green) and Hoechst stain (blue). Quantification of the intensity ratio of parkin in the nucleus relative to cytoplasm (right panel) indicates that parkin is excluded from the nucleus after SNOC exposure. Scale bar: 10 μm. Values are mean + SEM, n = 9 from triplicate experiments; * p < 0.01. B, Chromatin immunoprecipitation (ChIP) assay using a specific parkin antibody (MAB5512) and oligonucleotide probe encoding the human p53 promoter sequence. Control or parkin-overexpressing SH-SY5Y cells were exposed to 200 μM GSNO (or GSH) for 4 hours prior to performing the ChIP assay. Relative fold enrichment was assessed and normalized to control. Values are mean + SEM, n = 4–5; * p < 0.05. C, Electrophoretic mobility shift assay (EMSA) using recombinant parkin protein and oligonucleotide probe encoding the human p53 promoter sequence. Parkin proteins were pre-exposed to 200 μM old or fresh SNOC prior to incubation with the oligonucleotide probe. The optical density of shifted bands was quantified and expressed as percent control. Values are mean + SEM, n = 4; * p < 0.01.