Figure 3

NFκB binds to human PINK1 gene promoter. The effects of NFκB on the human PINK1 promoter in HEK293 cells (A), SH-SY5Y cells (B) and N2A cells (C) were analyzed by luciferase reporter assays. The PINK1 promoter reporter plasmid (pPINK1-A) or pGL3-promoter was co-transfected into cells with NFκB expression plasmid or the empty vector pMTF. Values indicate means ± SEM. n = 3, *p < 0.01 by Student’s t-test.(D) EMSA with NFκB p65 consensus probe. Lane 1 is labeled probe alone without protein extract. Incubation of the probe with NFκB p65 enriched nuclear extracts forms a shifted DNA-protein complex band (lane 2). Competition assays were performed by further adding different competition oligonucleotides including NFκB-mu, NFκB-wt, and PINK1-NFκB. (E) EMSA with PINK1 NFκB-1 probe. Lane1 is labelled IR700-PINK1-NFκB-1 probe only. Addition of NFκB p65 enriched nuclear extracts forms a shifted DNA-protein complex band (lane 2). Lane3 to 6, the competitor NFκB-wt and NFκB-mu were added at 10- or 100-fold. Lane 7 and 8, addition of anti-p65 antibody forms a super shifted DNA-protein-antibody band (lane7) and NFκB-wt was added for competition (lane 8).